Science topic

Cell Line - Science topic

Established cell cultures that have the potential to propagate indefinitely.
Questions related to Cell Line
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We recently acquired a CHO line from Sigma (00102307) adapted for growth in suspension in Sigma CHO Protein Free Medium. The cells are pink and not growing. Has anyone experienced the same?
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Did you check the pH of your medium make sure you have l-glutamine in your medium or if this fails add so hydrocortisone
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I purchased a brand new vial of Sf9 cells costing over £200 and thawed them exactly according to the recommendations. However, after 8 days they have still not grown at all - there is some loss of viability however the majority of the cells are still alive. They are cultured in Sf900II media with no serum as advised. Has anyone had a similar problem?
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Hi,
What was the volume for the SF900 which you added the cells into? They definitely don't like to be very dilute and if you dilute them below a certain cell concentration (less than 3x10 to the 5) it takes longer for them to come to a logarithmic phase. I start my SF9 cultures (fresh stock from the company) with 20-30 ml of SF900 medium with antibiotics (penicillin and Streptomycin) but without serum. Make sure that the medium is at room temperature before you add your cells into it. Thaw the cells in your hand (with gloves of course). Just add them into the flask with SF900 medium in it. I use 250ml flask with 20-30 ml medium in it. Check the cell number after 3-4 days. If they are growing fine and have a higher cell concentration dilute them to a cell concentration of 3x10 to the 5 by adding some fresh SF900 medium in it. Grow it further and for maintenance passage the cells every 3-4 days and dilute them whenever they are >2x10 to the 6 cells/ml.
If it is the case that they are very diluted and it looks like you might not want to buy another stock of cells : ) Then you can try to rescue the cells you have by centrifuging them aseptically in 50 ml falcon tubes at very low centrifugal speed (100xg) for 10 minutes. Pour off the supernatant and resuspend the cell pellet in a lower volume of fresh SF900 medium. Combine the resuspended cell pellets in a new flask and let them grow at 27 degrees 130 rpm. Check the cell concentration after 2-3 days and proceed as above.
This you might want to do it anyway because they like fresh medium after a while as there is toxic stuff accumulating after long periods of growth.
Hope this helps. You can see that if they are 'happy', they are round and shiny.
Good luck!
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Looking for cancer and normal cells, epithelial and fibroblast, which are also suitable for siRNA transfections. Any suggestions?
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what about Hep 3B and Hep G2???
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I suspect most such cells would be immune cell derived in some way, but it would be good to know what's out there.
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HL-60 promyelocytic leukemia cell line
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I have two questions. There are different available cell lines to create animal models of cancers. In the case of melanoma, there are different melanoma cell lines for instance melanoma B16F10 (metastatic), B16F0 (non-metastatic), which all derived from C57Bl/6 mice. Is there any data base for cell lines which explains each cell line in detail? May two closely related cell lines (e.g B16F10 and B16F0) differently response to a treatment?
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Yes, Vahid. There-se a cell line data base online where you can find information about those cell lines.
http://bioinformatics.istge.it/hypercldb/ is one of them. I also use B16F10, and I found information on that cell line there.
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I have problems with the ATCC’s distributor in Mexico. I ordered a normal breast cell line and it took eight months to send it. Does anyone know about another source of breast cancer cells? I need two triple negative breast cancer cell lines.
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There are several TNBC lines available from ATCC. You need to be aware of the fact that TNBC is a heterogeneous group of diseases. The cell line most often used is MDA-MB231. It is highly tumorigenic in mice, and metastatic subclones have been derived by Juan Massague`. However, this cell line carries a K-ras mutation (very rare in breast cancer) and is representative of the "claudin-low" subtype of TNBC. MDA-MB468 is a "basal-like" cell line which is PTEN -/- and represents the most common subtype of TNBC. Several other TNBC cell lines can be found in the ATCC catalog. For mouse "TNBC-like" cell lines, 4T1 is a good first choice.
mtRFP as a selective marker for stable cell line?
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I would like to create lentiviral derived transduced cell lines (neuronal and lung cancer) with overexpression and shRNA knockdown of my protein of interest. As I am going to study mitochondria I thought it would be a good idea to introduce mtRFP in the plasmid and use it as a selection marker for cell sorting. My questions are: 1. What promoters should I use ? ( I would like a strong overexpression of my gene). Will a weak or medium promoter before mtRFP give a signal strong enough for selection? Which promoters are not toxic and effective in both lung cancer and neuronal cells? 2.Is mtRFP overexpression toxic in some way or have any known impact on the function of mitochondria? 3. Are there any additional problems I should be aware during my design?
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I think that at least dendra2-mito is not very toxic to all kinds of tissue, because there is a mice, Pham-excised, expressing denadra-3-mito ubiquitously. It will be careful when you do long time observation, because photobleaching of fluorescent indicators releases lot of ROS. Roger Tsien has far-red fluorescence protein and images stems cells without any problem. Hope this works for you.
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We are studying this cell line and we have problems with the protein extractions because we realized that they are precipitated and we obtain few proteins. Which lysis buffer do you use for it? Any special previous treatment?
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Hi Jose,
The choice of lysis buffer depends on the cellular localization of the proteins you want to detect. A commonly used commercially available buffer is 'RIPA' which enables the extraction of membrane, nuclear and cytoplasmic proteins. If you want to separate cytosolic from nuclear proteins, a combination of two buffers should be used. I attached a protocol for you. Hope this helps.
Best,
Christian
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I am growing Sf9 cells in suspension culture and while they are growing ok, their viability has dropped considerably and so there is an accumulation of dead cell debris. Do I need to remove this? And if so any advice for doing so? Will the cells still grow ok with the debris and then I can dilute it out with subsequent passages?
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Preferably! Centrifuge cells at low speed. Dead cells will, hopefully, stay in the supernatant floating. Just discard supernatant and replate pelleted cells. You can check for cell viability.
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We are working on EA.hy 926 cell line from ATCC and we need to stimulate the oxidation of the cell line to produce more ROS, followed by using the dye DCF-DA or DHE. We tried TNF-alpha but it didn't work very well. Sometimes the control has higher fluorescence than the one exposed to TNF. Does anybody know what the reason may be, or if there is any other alternative way to stimulate the oxidation of the cell line?
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without knowing what you are looking at, there are quite a few options. One simple one is to modulate activity of the mitochondria. E.g. using antimyocin will increase ROS production in the cell.Another is to just add H2O2 to your solution and that will increase ROS.
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Two months ago we detected in our cell lines a contamination of mycoplasma using a Myco detection PCR kit. We tried to eliminate it: we threw away all cell lines, we clean incubators, wood, pipette, we have filtered medium, FBS and all the reagents but yesterday we had to repeat the test and cells are still contaminated.
Does somebody have experience with this problem and can suggest the correct way to identify it (also, if is possible, detect it with microscope) and definately eliminate mycoplasma?
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you could find some helpful information about the detection and elimination method of mycoplasma:
wish this may help you.
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I'm trying to get a hold of RMA-S cells. They are TAP deficient H2b lymphoma cell line that is used to detect peptide binding to mouse MHC. They seem to be widely used, but authors do not often say where they were obtained from, or they were obtained from another lab. How do I get my hands on them if I don't know these labs?
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I just tried to determine the origin of the "RMA" cells, but the literature from the 1980s about these cells does only roughly describe these cells as derived from "RBL-5" C57BL/6 tumor cells - but I could not get a clear answer.
Did you, in the meantime, get some "RMA-S" cells?
We have some "RMA" designated cells, but it remains to be shown what they actually are...
ATCC does not offer these cells, do you know why?
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I am using an insulin sensitising drug in adipose cell line. I need to measure the efficacy of it in improving the insulin sensitivity
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Hi,
You have different way to measure insulin sensitivity in cells.
You can measure the phosphorylation level of insulin receptor tyrosin residue or Serine 473 of AKT/PKB protein (commonly used in papers) or downstream protein present in the IR/IRS/AKT signalling pathway.
You can also measure glucose uptake in your adipose cells.
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I would like to quantify NO production from cell lines. I found a couple kits on the market and I am not sure what one to use. Does anyone have any experience using these kits. Can I use colored media?
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You can use Griess reagent assay which is based on colorimetric approach. To do that, you even don't need a kit and could prepare all the needed reagents in your lab. What you basically need is - 0.2% naphthylethylenediamine dihydrochloride, and 2% sulphanilamide in 5% phosphoric acid. And yes, you could use the colored media, like DMEM.
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I construct fusion gene stable cell (U2OS and MG63) lines by using lentivirus. But there is an absurd result. I can detec expression of protein (WB), but I cannot detect expression of mRNA (QRT-PCR). Why? Can anyone tell me?
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Are you using oligo dT for your reverse transcription?? Lentivirally-encoded transcripts are not polyadenylated. You need to use random hexamers or transcript-specific primers for the RT step.
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Electroporation could be used. Does anybody have an experience in electroporation with proteins or peptides? It would be very helpful if you share it. The second solution is streptolysin. Maybe there are any better methods?
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For proteins the following kit works....i dont know about peptides. But it may help you...
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Can anyone recommend one or more cell lines (primary or preferably immortalised) that can be used to examine neural spiking? References to protocols for observing/recording spiking behaviour would be greatly appreciated.
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I'm trying to do a stable transfection of hybridoma cell line NG108-15. I'm using FuGENE(R) 6 transfection reagent in ratios of 3:2 (reagent:plasmid) in 6 well plate and in confluence of 70%. After two weeks in G418 (250 ug/ml), all my cells are dead. Does anyone have any suggestions?
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I have three suggestions, the first is to titrate the G418 doses, the second will be to perform two sequential transfections in order to increase the efficiency of getting stably transfected cells and finally start the selection after only 72h. In ourt lab we are using Dreamfect Gold reagent for producing stably transfected cells.
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Some cell lines, for example Y79, grow in grape-like clusters. Is there any substance contained in the medium or FBS, or anything produced by these cells, which promotes gathering in clusters? Is there any way (except for physical force, like stirring) to prevent them from forming aggregates and make them grow separately?
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If you actually go to the ATCC web site page FOR Y79 CELLS (ATCC HTB-18) and not just an irrelevant generic protocol, as posted above, you will find that the subculturing procedure for Y79 does not involve trypsinization, since Y79 don't adhere significantly to plastic. The clumps are a normal feature of Y79 and you will see a photomicrograph of this at the same site. The ATCC subculturing protocol for Y79 is as follows (copied from the site and pasted here):
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:4 is recommended
Medium Renewal: Twice per week
Allow aggregates to settle to the bottom of the flask. Remove supernatant and discard. Add fresh medium, aspirate and dispense into new flasks.
THERE IS NO TRYPSINIZATION INVOLVED.
You can look it up yourself at:
You may have to register first (free), then under documentation go to PRODUCT SHEET and download it. You will also find references describing the cell line. Anybody using a cell line they are unfamiliar with should be doing this basic literature research before starting a project, not collecting uninformed opinions from people who have never seen the cells. That's just good science!
To more generally address your question: There are many reasons why cells might clump, ranging from divalent cations in the medium to cell adhesion molecules, serum contaminated with cross-linking antibodies, contamination with mycoplasma or fungi, etc. However, Y79 normally clump! Presumably they do not have adhesion molecules that allow attachment and spreading on plastic, but are able to adhere to each other. I have worked with CHO adhesion mutants (unable to attach to plastic because of fibronectin mutation) years ago that clumped in suspension. We selected non-clumpers by simply transferring the cells to a 15 ml conical centrifuge tube in 10 ml of medium and letting the clumps settle under gravity for 5 minutes, then taking the top 1 ml and subculturing that. After about 20 passages using exactly the same technique the cells were entirely a single-cell suspension. Unfortunately, the project was shelved without investigating what the difference was between the clumpers and non-clumpers. You may be able to do this with Y79. A WARNING however: this is a SELECTION process and whatever features of Y79 cells that are actually important to your work may also be altered as a result of the selection. Similarly, if when you subculture you discard loosely attached cells and only passage cells that are somewhat adherent to the plastic (and require trypsin to remove them) you will eventually end up with cells that are highly adherent to plastic. Researchers experienced with tissue culture are always wary of changing the properties of their cells by subtle selections due to technique and are always going back to frozen stock at regular intervals. The literature is replete with examples of cell lines that have diverged significantly in different labs because the methods for cell culture were different and put divergent selective pressure on the cells. Y79 are a (typical) karyotypically screwed up cancer cell line (hypertryploid with a lot of fragmentation anomalies) and would be VERY susceptible to such divergence.
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I have revived a vial of CHO PA DUKX parental cell line but it took 2 months to recover and then started growing fine. Now I suspect that the cells which I am growing are the not the original ones (because they recovered very tough and I think that there might be some genetic alterations in the cells). I want to confirm that they are from the CHO PA DUKX cell line only (the original without any mutations). So can anyone please suggest the type of test and protocol to perform to confirm this?
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Total Sequencing in this case if you are looking for specific/minor mutations.
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I need to check for IL-17 production by FACS on primary human cells and I would like to include a positive control on my assay. Human primary Th17 cells are quite tedious to isolate; I need something more straightforward (if possible...). So, does anyone knows a cell line expressing IL-17 upon stimulation?
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I realize this answer comes late but maybe you can generate your own positive control and freeze these cells down. There are many protocols out there to generate short-term human T cells producing IL-17A
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From primary cells cultures of the human pancreas, we are getting positive immunostaining of cells for anti-insulin and anti-glucagon. We also get positive results from RT-PCR using a primer for insulin. I would feel better about our results if we had a definite positive control. I'd appreciate any comments at all about this.
I'm using an entire pancreas as a positive control, but I think that a cell line would be more comparable. Also, is there a human organ that would be absolutely negative for insulin?
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Pancreatic Beta Cell Lines and their
Applications in Diabetes Mellitus Research
Masa Skelin , Marjan Rupnik and Avrelija Cen
Please read the article to find a table on insulin positive cell lines.
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I'm trying to create a cisplatin resistant cancer cell line, HCT-116. Does anyone know the optimal concentration that I must add to the culture? I started with 1μg/ml of CDDP. I noticed that the drug killed most of the cells, but the levels of ERCC1 have decreased.
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Steps:
* Determine IC50 and IC90 dose of drug for 72h treatment.
* Treat cells with IC90 dose for 24h.
* Replace the media with fresh media containing IC50 dose and maintain it.
(The shock will kill most of the cells.)
* Some cells (less sensitive to drug) will remain alive for long time. Maintain the cells in that concentration unless it start division.
* Once confluent maintain the cells at the same dose for at least 5 generations.
* Then increase 1.5 times of the dose and repeat the same.
* Keep on increasing dose slowly for some time (till 4-5 times of IC resistance dose not come). Once cell will reach that conc, it will start expressing its resistant proteins in a stable fashion.
* Then increase the conc. twice or even thrice the previous dose, and cell will remain viable.
Note: 1) Make stock at every stable stage (after stable resistance at that dose).
2) Addition of glutamate (natural autophagy inhibitor) in the media may help some time.
3) If cell size increases (twice of the normal size) do not use that cells and start with a lower dose and with fresh stock. Some increase of cell size is expected.
G-banded karyogram image of BHK-21 cells
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Does anybody have an image of a G-banded karyogram of BHK-21 cells? It is almost impossible to come across one in the literature nowadays. Thans in advance, Vasilis
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Hi Vasilis, I see, so you have a light microscope for analysis, but not the equipment to capture the image and create a karyogram? Is that correct? metaphase images would be enough to include in a paper if they showed the chromosomal alteration you are studying. If you cannot capture an image, it would be prudent to get someone to create an image from your cell line, rather than relying on another derivation of the cell line from another centre. Good luck
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Would be interested to learn from someone's experience
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Bacterial lipopolysaccharides (LPS) activate cells of innate immunity, such as macrophages, by stimulating signaling through toll-like receptor 4 (TLR4). It has been hypothesized that LPS derived from different bacterial species may function through TLR4-independent mechanisms. To test this hypothesis, you can generate using a nonviral transformation procedure a bone marrow-derived macrophage cell line called 10ScNCr/23 from mouse strain. C57BL/10ScNCr. This mouse strain has a deletion of the TLR4 locus, causing the mouse strain to be nonresponsive to stimulation by LPS from Escherichia coli while responding normally to other bacterial substrates, such as lipoteichoic acid (LTA) from Staphylococcus aureus, which signal TLR4 independently.
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I am using silver nanoparticles, 20 wt. % (dispersion in organic solvents) (sigma, product # 719048) for an MTT assay on a HL-60 cell line and I am not getting what should be my vehicle control?
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Shouldn't vehicle control be organic solvent speaking in general as thats what u dispersed test item in? am not sure about purchased nanoparticles but gen if you make nanoparticles in water then that water and reactant solutions are controls (to my knowledge). Also, can you please elaborate when u said "am not getting"? Am wondering if all treated cells were dead or were the NPs seem to be non toxic?
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I'm trying to assess the utility of various in vitro assays/models, and I'm looking for *general thoughts/opinions* here. Elsewhere, I'm looking at the specifics of modelling the brain, modelling nanmomaterial-blood interactions, modelling cell transplantation etc.
I suspect each aspect of each model would need to be tested, and then the data compared to whatever in vivo data is available (if any; it may be incomplete, or rely on surrogate outcomes/biomarkers).
E.g. for a white blood cell (WBC) in vitro model: are WBC responses to immunosuppressive agents in vitro, identical to responses in vivo? You'd presumably have to measure the levels of lots of cytokines, in vitro and in vivo, and some may correlate better than others. (also morphological, motility, toxicological assays, etc.)
I presume that you can only conclude that (this specific) in vitro WBC assay is a good model of (this specific) cytokine release in response to (this specific) immunosuppressive agent.
Strictly, you shouldn't extrapolate from this to assume that, say, apoptosis or chemotaxis in this model would also be representative of in vivo, but are there any standardised approaches to this kind of vitro/vivo comparison?
Have any rules for generalisation/extrapolation emerged? (e.g. if the in vitro secretome of a model is representative of in vivo, can we expect proliferation or apoptosis to also be representative of in vivo? Or is every element distinct?)
I'm sure most models work well for some aspects, but not others (e.g. rodent models vs human). Is there any database where failed/predictive applications of models are reported/collected?
Is there any database comparing (specific) cell lines with primary cells and/or in vivo data, for various assays?
I appreciate that this is broad and woolly, but I thank you for any comments on this.
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The validation of in vitro models to "mimic" in vivo ones is a really difficult issue. In general is assumed that for a specific process studied (for example eye irritation, skin irritation, corrosion, contact allergy, and so) an only in vitro model would probably not be sufficient.
In Europe the European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM; http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam) is the organism that receive proposed alternative methods which reduce, refine or replace the use of animals for safety testing and efficacy/potency testing of chemicals, biologicals and vaccines. This validation process implies several steps and different organisms and last several years. In USA the responsible is ICCVAM (The Interagency Coordinating Committee on the Validation of Alternative Methods, http://iccvam.niehs.nih.gov/). Both organisms have databases with the protocols of validated methods, Advisory and Consultation Bodies, comments and recommendations. Another interesting site is AltTox. Org (http://www.alttox.org/about/), devoted to the development of alternative methods to toxicological studies.
When developing an in vitro model this should be mechanistically based on the in vivo process, scientifically supported and based on well-known responses described in vivo in front of a battery of chemicals and/or products. The comparison of responses in vivo and in vitro is a crucial parameter and ideally the in vitro data should be compared with human data. However, comparisons are generally performed with the results obtained using animal models.
As you see, the development of in vitro models is not a mere question and for this reason very few assays have been nowadays being validated and adopted. On the other side, it is an interesting way to deep into the physiological, pharmacological and toxicological mechanisms and thus to increase our scientific knowledge.
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1301 cell line doubling time.
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FYI: In my recent experience, the doubling time of 1301 cells in suboptimal expansion conditions of continuous agitation (ie with my own hands) is 3.5 days. Oh my god! my fortune for a spinner flask for growing cells in suspension...
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Stable cell line.
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If you want to transient transfect and select, I think it is more dependent on the cell line you use. A good selection marker (puro / G418- whichever works well in the target cell line) and not so bulky size would be 2 features necessay for the plasmid (In addition to the promoter criteria mentioned above- in case of not well tolerated proteins).
But as an exception, I have made several cell lines with CMV promoter expressing plasmids (strong promoter) and have also selected cell lines by co-transfecting resistance containing plasmids separately. At the end of the day, it depends more on the toxicity of your expressed protein and the tolerance range of the cell line..
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I am trying to make a stable cell line that expresses my protein of interest. I have the gene of my protein in a vector that provides puromycin resistant, but I am not sure what protocol to use. Should I just transfect the DNA and then do the selection with the adequate concentration of antibiotic?
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Dear Edna,
Here are my protocols:
Day 0: Seed target cells in 24-well plates. The number of seeding cells differs according to the cell proliferation rate.
Day 1: Transfect cells with virus. For polybrene accessible cells, mix the culture medium with proper concentrations of polybrene. Replace the medium completely with 0.5 ml polybrene-containing medium. For polybrene sensitive cells, this step can be skipped.
Day 2: Refresh the culture medium 24 hours post infection.
Day 3: Change to fresh medium with puromycin 48 hours post infection. The recommended concentration of puromycin ranges from 1 to 10μg/ml according to cell lines.
Additionally, the killing dose of puromycin vary from cell lines, 0.5-5 ug/mL in common. First, I suggest you use a couple of wells to titer the concentration for each cell line to find the optimal dose. The cells may die after screening for 2-3 days. Second, it works well to screen with puromycin when passaging.
Genemedi got a rich experience in lentivirus production and transfection, you could find more information on http://www.genemedi.net/i/lentivirus-packaging
Hope this may help you.
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I want to measure changes in intracellular calcium levels in BV-2 cells. Does anyone have experience with LPS and its effect on calcium in this microglial cell line? I tried to measure it with Fluo 4 after 24 hours exposure and there was no increase compared to my untreated control. Thank you.
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I haven't tried calcium measurement on this cell line. However, I have done a lot of calcium expe on Hela cells with Fura-2. I prefer to use Fura-2 since it's represented by the ratio of F340/F380. I think you could firstly apply a positive control drug, which could trigger obvious intracellular Ca2+ increase, to see if your experiment set up is OK.
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Seeking KYSE SERIES OR TE series cell lines
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Applied Biological Materials (ABM) has a huge cell line collection. You can try them. (www.abmgood.com)
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We want to transduce T-and B-lymphocyte cell lines but transduction efficiencies are poor, i.e. western blots are negative but RT-PCR is positive.
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No, years ago I worked, with the gateway system, and setup things for actually using lentiviral system fiddling around with hybridoma's but I actually wonder if in the end I used it, or that an alternative gave quicker results. But if even GFP is expressed poorly, this must mean that indeed either the promotor is not ideal or more likely you hit indeed too little. I looked quickly to my protocols, I only have protocol for adherent cells wich is probably not much of a help.
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I am working on Hamster buccal pouch model and I would like to do in vitro studies. Can anyone suggest a good cell line for my studies?
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Would you be OK to work with human and mouse Oral Squamous cell carcinoma cell lines, too?
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I need to start a stable cell line using HEK293T cells, antibiotics, serial dilution and random integration over time. I have never seen the procedure but I read about it, however it is still quite vague. Does anyone have any videos or further information regarding this procedure?
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I've done this several times with transfected cells and I think the reason it's usually vague is that there's not a whole lot more to it other than adding the appropriate concentration of antibiotic and waiting. I'll give you a couple tips I've learned from my own experience though:
1) Never let the cells reach confluence while you are selecting for transfected cells. You want to keep them all rapidly dividing to give the growth advantage to cells expressing your gene. If you're using a serial dilution, then you'll likely find a good dilution after transfection where the cells are not too confluent too soon after beginning treatment.
2) After exposing the cells to antibiotic for 48 h (the time needed for normal cells to double twice in my case), I would thoroughly trypsinize the cells, inactivate the trypsin, and let the cells re-attach in the same flask for 4-5h, then wash off the weakly attached or non-attached cells. The idea is that non-expressing cells can double twice in the presence of antibiotic but after that, they can not make new protein (in the case of G418), so they will not be able to re-attach.
3) Be sure to change your media regularly, even if the cells have not used up all the nutrients, the antibiotic will likely be inactivated after a couple days.
Hope that helps.
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I recently I needed to perform a rescue experiment for my gene. Since my stable shRNA cell line seems a little bit weird, I want to switch to generate a stable overexpression cell line first, then conduct knockdown by siRNA. However, I still feel not quite confident to do that, since most people will use shRNA+transient OE or coexpress siRNA+OE. Could this stable OE+siRNA OK? Could anyone show me some publications as examples? Thanks!
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Hi Candy,
I wanted to try the transient knock down and co-transfect. Did you go ahead with the above protocol? I understand it's been a long time and you might not recall details of the experiment but can you share your results and any comments that you may have after doing this experiment, whatever you can recall?.. Thanks for your help!
Pallevi
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Is there any database where I can check what kind of surface receptors are expressed in different cell lines? Interested to compare two cell lines A431 and HEKn to see if they can be identified by receptors that are expressed on one but not on the other. If there is a source with all listed receptors for different cell lines would be nice to hear about it. Thank you.
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Could someone shed light on what happens if cells are kept for more than 5 minutes in 0.25% trypsin and why?
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I am working in a repository of fish cell lines. Some of the cells are rigid to detach. I have to keep more than 5 minutes in 0.25% trypsin. Is there any harm to the cells occurs through this.
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Trypsin is a proteilytic enzyme and used in a particular concentration for particular time to digest cementing material (extracellular matrix) to detach the cells. If we use trypsin for more than that it will start digesting cellular plasmamembrane protein also. and damage it. The serum used in culture media has antitrypsin activity and as soon as we add culture media to the cells trypsin activity is stopped. hope your query is answered.
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I am just wondering what might have caused this as this is happening many number of times and also in 12 wells and 6 wells plates. I know it might be because of edge effect/evaporation but the medium volume looks very much the same in all wells but i could see a change in color on the side wells. Incubator we use is a new one and everything seems to be fine. How to rectify these problems or else any other alternatives. greatly appreciate your answers.
thanks,
durai
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agree with Saori..
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I was trying to find the catalog number of this cell line and reliable source from where I can purchase it. Could any of you please help me provide this information regarding HiB5?
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This is second paper you can use as reference.
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I am looking for a syngeneic immunocompetent mouse model of OvCa (in balb/c or C57BL/6J, preferentially IP).
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From Dr. Katherine Roby at the University of Kansas Medical Center. Her Email address is kroby@kumc.edu.
Viji Shridhar
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ARPE-19 cells are retinal epithelial cell line that can get differentiated when fed with low levels of serum (1%) for at least two months.
After the differentiation, they loose the ability to replicate and they are more similar to the in vivo condition. Since they cannot replicate, I really don't know how to store them. I tried to freeze them as usual by using FBS+ 1% DMSO, store them in -80°C and then seed them again: in this case, some of them attached to the petri, but a larger part died. Can you help me? I would appreciate any advice from you all to escape this situation! Thank you!
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Ciao ragazze..alcune de voi ha visto le Arpe-19 confluenti ma con una capa di cellule redonde (round) che proloferano molitisomo!? Io uso dmem/f13 10%sfb
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They formed nice colonies and tolerated well for G418 when I was selecting.
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The main problem with transfection is that of where is your plasmid! Sometimes it is maintained as an episomal plasmid like structure, and persists for only as long as your selection. The alternative is that it undergoes a recombination event to enter the genome. If this is the case and your gene of interest represents 20-30% of your plasmid, then it has the same probability of being cut up and therefore will be inactive. Generally speaking I will screen a population of clones and find a wide range of expression of the target gene, most of which will be low. After screening I freeze down as many vials as possible. I then only use the cells for a fixed number of passages, then disgard them. I thaw fresh cells, grow them under selection, remove selection for a passage or a week (whichever is longer) then do the experiment, which will include an arm of the experiment to determine whether expression is still what it should be.
G418 can be tricky as it is quite slow to act, and if your cells are growing fast the cells at the centre of a colony will not be exposed to as high a concentration as those on the periphery. I usually add G418 during passage to ensure that all the survivors are actually resistant.
One strategy is to digest your plasmid with an enzyme which linearises the plasmid in a region that does not contain your Neo or gene of intertrest and then transfect again, but you need much more DNA as it doesn't last as long as it is single stranded and not a closed circle. This has a greater chance of any recombination events not occurring within your selection or gene of interest. An alternative is to cut out your gene of interest and paste it into a retrovirus, and infect your cells. Also if you can use one which contains puromycin resistance gene as the selection is much quicker.
Good luck!
What is a good way to check whether a receptor is present or not in newly established cell line?
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I want to check in a new established cell line whether a receptor is present or not ? How to go for that.
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Hi Amit, if there is an antibody against the receptor available perform a staining protocol for FACS analysis. If you have no access to a FACS you can try an immunfluorescence staining and analyse your cells via fluorescence microscope. Or you perform a Western Blot analysis, but this gives no spatial information on the occurence of your receptor until you isolate the specific fraction (i guess in your case the membrane fraction). Best wishes, Guido.
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We are working on muscle nAchR and have plasmids for 4 subunits i.e. alpha subtype 1, beta subtype 1, epsilon and delta. In addition to this we have a CFP plasmid. Untill now we were doing transient transfection in HEK-293, but now we want to have a stable cell line. Is it possible to have stable cell line with all five?
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We have generated such stable cell line with the AchR complex from a sea snail. It took us 3 weeks to derive the line. We made use of a FACS based strategy with a labelled ligand to isolate active clones.
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I found many protocols using FGF treatment for lens epithelial explants, but i have found only one differentiation protocol for this cell line, using serum-free DMEM/F12 medium supplemented with insulin, transferrin, and selenium. I was expecting to find at least a protocol using FGF or BMP which are the main effectors in lens differentiation. Maybe these cells don't respond well to this factors? Thank you.
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perhaps it depends on cell lines, in my case cells from BL6 (7Weeks) did not attache a surface without serum and i used always FGF2.
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The cell lines are T24 (bladder cells) and bacteria is UPEC 2980, one of my extract block the cell receptors and the other bacteria side, which one is better for using in vivo?
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I am considering cell-cycle-synchronising a slow-dividing cell line (say, 1 week doubling time). Has anyone had any relevant experience? In particular, any suggestions how to relate the more-or-less standard procedure of synchronising fast cells (e.g. HeLa, MCF-7) by a double thymidine block (16-24 hours on, 8 hours off, 16-24 hours on) to the doubling time?
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Thanks, Rustem - I will definitely have a look!
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NKG2D is a receptor expressed on NK and T cells. I would be grateful if anybody could suggest a suitable cell line that can express NKG2D on its surface for in vitro studies.
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YAC and CHO cell lines
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I recently switched from Human Recombinant Insulin to Insulin from bovine pancreas and my MCF-10A cells start to look weird after a few days in culture. They don't become 100% confluent, tend to be patchy and not the characteristic "cobble-stone" like appearance that they should have. Anyone know if this could be a result of the change of insulin or any other ideas?
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I changed a few things in my media but I don't think it was the insulin because it should work with both human and bovine insulin. Try making all your ingredients fresh and see if you can get a new vial from another lab that is at a low passage number. This worked for me. Hope this helps.
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Isolation of cell cultures is only possible in 10% of the cases. Most tumor samples do not show outhgrowth. Does anyone know why? Are there studies on the involvment of certain mutations within the native tumor?
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Collectin of fresh material into a transport medium is essential to keep the cells in condition prior to disaggregation and plating. SOme lines prefer to establsih out of small clumps rather than single cells so try both ways. When I was establishing Merkel cell carcinomas, I found that putitng them in the incubator and leaving them alone for a few days was beneficial. Too much movement in and out of the incubator to see whether anything is growing stops cells settling and disturbs any microenvironments being established. These are often beneficial if you are not sure what added supplements are required. Good quality medium and serum is required. Use of irradiated feeder layers of fibroblasts or endothelial cells can help depending on the type of cells you need to establish.
Also if you are not having much luck try out some of the different plastics for culture dishes. Just changing brands can work, but there are also ones now with specific coatings on the surface which assist various cell types to establish.
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I have isolated total RNA from u2os (human) cell line. All times my 28 RNA band was not sharp from 18s RNA it got degraded. Anyone have suggestions?
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If you are using kit please follow striclty the manufacturer's recommendations. And try to be as quick and effecient in executing the protocol as you can. Make sure that all your micropipettes are deignated for work with RNA isolation only and all your filter tip, microfuge tubes, plastic and glasswares are RNase-free. Treat your bench area with RNasedeactivating solutions first such as EDTA/NaOH follwoed by DEPC-treated water prepared at a final conc of 0.1% and had been autoclaved. Always minimize the time between sample collection and addition of lysis buffer. Better to work with fresh samples and if you have to stores your tissues you must snap-freeze them in liquid N2 as quickly as you can and store them at -80°C untill further use. You might alternatively dip your tissues in RNA-inhibiting solutions such as RNALater which is quite effective and you can store it as such in the normal fridge for a few days before you actually extract them. If you are working with a traditional way make sure that all your reagents autoclaved, filter-sterilized, and prepared using DEPC-treated water and all other roles applies and always work on ice until you finally elute or suspend your isolated RNA in buffer or water. Best o luck. mourad
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I am looking for human diploid immortalized cell lines, that are well established. They should be easy to culture, to transfect and also grow at low density to allow the selection of clones.
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I was looking for the same type of cell lines and I came up with the RPE-1 cells. They are hTERT-immortalized cells that maintain a diploid karyotype (46, XX, der(X)) in 90% of cells. According with ATCC, they are also good for transfection. I definitely will give them a chance.
I hope it helps you.
Oscar
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We are going to study secondary messenger antagonist effects in respect to calcium mobilisation in a pancreatic beta cell specific cell line i.e. Pancreatic beta MIN6 cell line
I also want to know about specific media for sertoli cell derived lines.... MSC-1, GC-2 spd, SF7, 15P1 etc.
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Ok, thanks a lot Shiv Mishra!
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PS is a pig kidney cell line used for titration of flaviviruses. I require an early passage PS cell line, if anyone has it? Or the source from where it can be procured.
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Yoc can check NCCS, Pune and IMTECH Chandigarh.
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for my research I need mouse glioma cell line (GL261 or GL26) but i cant find this cell line.! in some paper i see that this cell can be prepared from ATCC but no information can be seen in the ATCC website.
if anyone know how i can find this cell line, please help me.
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In my thesis I've worked with GL261 mouse glioma cells. In the links below you can find the catalog and the ordering information from the NCI.
Good luck!
Karyotype study of a human diploid cell line
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My question involves the rational involved in a karyotype study for the characterization of a human diploid cell line or a human stem cell line. To be more specific I will express my queries in bullets: 1) Do the same rules of clonality apply as stated by the ISCN for neoplasia? 2) Are 20 metaphases enough for the karyotype study at any given passage or 50 metaphases is standard? 3) If a non-clonal aberration is detected during the analysis (20 or 50 metaphses), should it be mentioned in the report? How many more metaphases do you think are "adequate" to draw a conclusion? In case more metaphases are studied and the aberration remains non-clonal then should it be mentioned in the karyotype report?
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(1). same rules of clonality as indicated by the ISCN would apply. (2) I would do 20 metaphases - I think this is sufficient for a study. (3) If non-clonal aberration is detected, scan for abnormality in 10 more cells. If it is a characteristic aberration associated with any neoplasm, I would increase the count. Many times if it is characteristic it will be there. A non-clonality cell would be mention in a report for future reference only. Rolando.
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Does anyone know any cell line containing integrated HPV16 genome?
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SiHa (ATCC # HTB-35) contains ~3 integrated HPV16 copies per cell.
CaSki (ATCC # CRL-1550) contains ~600 integrated HPV16 copies along with some HPV18-related sequences per cell.
Typica control: C33A (ATCC # HTB-31) is a cervical cancer cell line that does not contain any HPV copies.
Hope that helps, if you have any other questions let me know.
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I am conducting an a549 cell line transfection. I used FuGENE HD for transfection and I am only getting 30% effeciency. What is going wrong? Is there a better agent I can use? If so, which?
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30% efficiency is really low. We make a transfection reagent designed for A549 (http://altogen.com/product/a549-transfection-reagent-lung-carcinoma-ccl-185/) and we've achieved upwards of 80% efficiency. Since A549 is a lung cancer cell line, there are some ways to optimize transfection by including cell-specific targets and using complex condensers, all of which are included in the kit above.
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It seems very difficult to find a good sertoli cell line to work with, any suggestions?
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Thanks. I am currently trying to obtain a sertoli primary culture......
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How can commercially produced recombinant Epo in CHO Cell line be analyzed for bio-activity or bio availability?
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EPO can be assayed by proliferation assays using EPO sensitive cell lines - NFS-60 ( murine) or TF-1 (Human).
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I am looking for human cell lines of myeloid or lymphoid lineages which are immortalized but not derived from tumors such as leukemias or lymphomas. Can anyone help me with this? Moreover, since I'd like to stimulate these cells with IL-2 and IL-6, they should express the respective receptors.
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EBV-infected LCLs (lymphoblastoid cell lines) in general express IL6R and may respond to exogenous IL6. These are not tumor-derived but generated by in vitro infection of human B lymphocytes with Esptein-Barr virus.
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When culturing THP-1 cells, some papers reported to use heated-inactivated FBS while some others use non heated-inactivated FBS. According to ATCC protocol, it seems that antibiotics are not recommended for the culture of THP-1 cells, but in most published papers, both penicillin and streptomycin were used for the culturing of this cell line. Are there any differences in THP-1 cell growth and differentiation under these different conditions?
Recently, we tried to thaw our frozen stocks of THP-1 cells, but most of the cells tend to become adherent. Anybody know what's wrong with the cells?
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THP-1 grow perfectly well in normal RPMI +10% FCS (non heat inactivated, we use Hyclone) antibiotics are optional but well tolerated! Adherence is a sign of suboptimal culture conditions and should disappear within a few days in correct medium. If not get fresh cells maybe your cells have been simply mislabeled.
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You read about how people realise too late that their cell line has been contaminated by, for example, HeLa cells which outgrow and overtake the original cell line. So what is the best way of confirming that a cell line you are working with now, is the same one you got a the start of your project?
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Whilst your comments are relevant, I would recommend against getting a sample from another researcher, as the same problems may occur. If you can get another vial from ATCC or DSMZ, then this is a viable option, but you may have performed a lot of lengthy experiments on the cells you have already. Therefore acquiring additional cells may not be tenable, or at least could result in a huge amount of additional work to characterize your new cells for whatever phenotype you are studying. If this is the case, then the primary focus will be to establish the origin of the cells you are working on.
Furthermore, your point about clonal evolution is valid, but is it really an issue? PC3 has an incredibly rearranged genome and that level of rearrangement is almost never seem in the context of primary prostate cancer. The cell line was derived from an aggressive tumour and has been established for many years. In general, cancer cell lines by definition have acquired many properties (such as DNA and chromosomal lesions) that provide an in vitro growth advantage and the genome will have evolved significantly since the cell line was established. There is the long-standing question of how accurately cell lines reflect human disease? Any given cell line may have an important biological/ cellular phenotype worthy of study, but whether that cell line reflects the pathogenesis of the derived tumour is in question.
Therefore, the biological properties of a cell line are likely to the critical research issue, so you have to wonder how relevant any degree of clonal evolution is, particularly in the context of a system defined by genomic instability and clonal evolution. That said, this is a long standing and contentious question, and many individuals will have different and equally relevant views on the subject.
However, even if you might consider clonal evolution to be relevant, it is highly unlikely that STR analysis (at least in the context of the commercial assays available) will provide meaningful insight into clonal evolution. Depending on the level of contamination, the same could be said for the use of STR analysis in this context.
Cytogenetics or molecular cytogenetics may not be appropriate in all situations, but to suggest that it cannot measure chromosomal heterogeneity between cells is incorrect. After all, it is a single cell approach (with the exception of CGH approaches). Of course, there are issues of sensitivity, particularly in light of modern technologies, but one of the premises of the clinical application of cytogenetics has been to identify genetic heterogeneity, in the context of cancer and constitutional disease. Examples would be mosaic aneuploidy, maternal contamination of prenatal samples, and clonal evolution of haematological malignancies. The entire purpose of the development of Hooks tables was to exclude levels of chromosomal mosaicism.
Of course, to exclude low levels of contamination or subtle clonal evolution would be beyond the realistic limits of cytogenetic or molecular cytogenetic approaches, even with the design of informative FISH probes for interphase analysis.
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I'm planning to start designing an in vivo assay to be used for a HTS. Does anyone have any advice/know of literature on the subject on how to choose an appropriate cells line? I will have to establish a stably transfected cells line with at least two constructs. The readout will be measuring changes in GFP expression.
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Dear Mrs. Bobrowska, I believe there is no right recipe for that, but it will really depend on the endpoint or biomarker that you are interested in. In your case, as you are transfecting a cell line with two constructs, I would suggest you to look for references on transfection rather than on HTS. I will try to contact some people that work with transfection to check if I can assist you any further.
Best regards,
Patricia
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I've been trying to analyze CoQ10 levels in cell lines +/- treatment with oxidative stress-inducing agents. I've tried methanol/hexane, ethanol/hexane and isopropanol hexane extraction from 5x10^6 cells, followed by drying down in a speedyvac and resuspension in ethanol/methanol FeCL3 prior to reverse phase HPLC (using Ethanol:Methanol:Perchloric acid mobile phase). However, I don't see any peaks corresponding to that obtained with control preparations of CoQ10. What am I doing wrong? Any help would be much appreciated.
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We normally use a single dilution step extracting 50 ul of a cellular suspension with 250 ul isopropanol. after centrifugation we inject directly and with the ECD method we are able to quantify as low as 1x10^5 cells
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MC3T3-E1 is an Osteoblast cell line working as a primary cell
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When do you need them, and how would you pay for them to travel from Australia to Lucknow? My lab is about to bring them back up from frozen stocks over the southern summer. What import problems will you have regarding customs?
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MDCK is good morphology condtion in DMEM when passaged in our lab shows slightly different and bigger in DMEM prepared by us. Its difficult to find out why. We don't use CO2 during incubation and is it making this difference?
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MDCK cell line can go further into several passages and they are very strong cells compared with other types of cell culture like PHTBE cells .... If you do not use 5 % co2 you must add HEPES to the media (1/100 dilution) ..... also try to use DMEM gibco 500 ml bottle with addition of 5 ml pen/strept and 5 ml HEPES and 0.5 ml Amphotrocin B and 10% FBS ...... that is it ..... I did notice bigger size before when I used antibiotics several times to cells to retrieve them from bacterial contamination ... the cells were so weared take long time to grow and show bigger shape ... so either you do not have the correct media or have bacterial contamination or used too much antibiotics that is toxic to cells ....
How to isolate/pick single cells in order to make a isogenic cell line?
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I wish to create a isogenic cell line with TALEN technology, but have had some problems in picking single cells from a culture of transfected cells in order to establish a pure cell line. Do you have any suggestions as to how this can be achieved?
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I have used the whatman technique myself. You can cut little circles with an standard hole puncher and then sterilize them by autoclave. I used to leave whatman soaked in trypsin for only 3 min, some cells will remain attached but you will pick up more than enough for your passage.
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Recently, I have found there was no CPE shown on c/36 cell line after infection with DENV2. I have seen the CPE on that cell line before. Is it possibly because of the over passage of the cell line? Or a virus problem?
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I got the same problem with the high passaged Dengue virus stock. Not only with C6/36 but also with BHK21. Thus, in order to determine the titration of virus, I needed to employ other techniques such as FFU ( focus forming unit) or FFFU fluorescent focus forming unit on VE6.
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We would like to screen an RNAi library for metastasis players in human samples but not sure which cell line or assay should be used.
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In our commercial xenografts, we've noticed that MDA-MB-231 is one of the more aggressive cancer cell lines with metastatic potential (http://altogenlabs.com/xenograft-models/breast-cancer-xenograft/mda-mb-231-xenograft-model/). Although we do try to maintain only one tumor for statistical purposes, metastasis studies are also possible and are easily done with such a well-characterized cell line.
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I need them as a negative control in my experiment for localizing TAARs on thyroid epithelial cell lines.
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Citation: "When we isolated a number of immune cell types from mouse spleen and assayed for Taar mRNA, we found that although spleen and T cells were negative for expression of Taar genes, B cells and NK cells showed expression of several Taar mRNAs."
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I want to use an aged cell line. Does anyone know how to make a cell line aged quickly?
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This paper may be of interest
Methods Mol Biol. Author manuscript; available in PMC 2008 June 9.
Published in final edited form as:
Methods Mol Biol. 2007
Aging Cell Culture
Methods and Observations
Sharla M. O. Phipps, Joel B. Berletch, Lucy G. Andrews, and Trygve O. Tollefsbol
Culturing and subcultivation of normal human diploid fibroblasts have advanced our understanding of the molecular events involved in aging. This progress is leading to the development of therapies that slow or ablate the adverse physiological and pathological changes associated with aging. It has been established that normal human diploid fibro-blasts can proliferate in culture for only finite periods of time. Hayflick and Moorhead and others have described numerous types of cells, ranging from fetal to adult, that were incapable of indefinite proliferation. There are many ways to study aging in vitro, and this chapter summarizes some laboratory procedures.
Good luck
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I need a cell line to express proteins that are under control of the T7 promoter.
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E.coli BL21(DE3) is absolutely a good choice!
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First, I got the cell line from ATCC and thawed it and seeded in DMEM medium with 10% horse serum and the cells gradually died. I then bought another vial and I split them in two different media, one is DMEM with 10% horse serum and the other one is DMEM with 10% FBS. Neither of them worked. I then requested one vial from another lab and thawed it and grew it in DMEM with 10% FBS and the cells grew very well. I froze some in 5% DMSO. A couple of days ago, I thawed one vial and the cells didn't grow, and I noticed it started to die like the one bought from ATCC. I was wondering what happened with the cells. I can't figure it out.
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Dear Xue Su, DMEM contains higher concentrations of aminoacids and glucose. So, you can grow EL4 in this medium up to some more cell density without outgrowing.
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My research is on expression profiling of microRNA in acute promyelocytic leukemia. I need the NB4 cell line as my positive control.
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We have it - you should be able to get a vial from DKFZ or similar?
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I am looking for a lymphocyte cell line that supports the development of merozoites and a tick cell line for the support of sporozoites of theileria.
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No problem, glad I could help.
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I'm working with culture medium RPMI 1640 incomplete, only being added antibiotic, and do not know if freezing can change some of the same property.
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This answer is a little late, but it may help someone:
According to ATCC, freezing growth medium (esp below -70) may cause the growth factors and/or vitamins to precipitate out of solution. I have also read elsewhere that -20 freezing destabilized some of the amino acids in RPMI, but I can't confirm that.
Here's a link to the ATCC cell culture tech doc. See page 16 for the relevant info.
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I want to infect a human cell line using a pSIN expressing vector but I do not know which packaging plasmids should be used with this vector and which proportion of each plasmid is optimal for packaging.
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Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA. Science. 2007 Nov 20. ():. 10.1126/science.1151526 PubMed 18029452
DEPOSITOR COMMENTS
The psPAX2 packaging plasmid and pMD2.G envelope plasmid can be used with this vector.
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I am using EML cells: a cell line derived from murine lymphohematopoietic progenitors immortalized. They grow in IMDM + 20% FBS + Sodium bicarbonate and 200ng/mL SCF.
They grow nicely for 2 weeks and they start to die and differentiate. I change the medium every 2 days. I centrifuge them at low speed (300g or 7 mins) and resuspend them in fresh medium. I have tried different concentrations (50000, 100000, 200000 or 300000/mL) but I haven't seen any difference in their growth. I always prewarm the medium at 37C to reach the correct pH.
3 people in my lab have tried to grow them and we are all experiencing the same issues.
If anyone has experience with these cells and can give me some advice, It would be really appreciated!
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There are several people who had had trouble culturing these cells. Where did you get your line from? Have you tried using SCF from BHK/MKL cells? Do they have the classic "hand-mirror? shape? Do you spin them every time you expand them?
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I want to construct a new cell line from tumorous cells.
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This is a very open ended question, so I will try and give definitions for my answers. Firstly in the case of purifying growing the cells over the time period of the experiment one does not have to worry to much as this is really a primary culture and one does not need to consider the continued proliferation of the cells. Katrin makes a really good suggestion, and If I were doing the experiment I would adopt that strategy. However a cell line is a different proposition. Most cells in primary culture will proliferate until the reach their Hayflick limit, which is a point at which they will no longer grow. This will generally be not much more 60 cell divisions, depending on the initial source of the cells etc. This is in part determined by the length of the telomeres in your initial isolation. If your goal is to generate a stable and immortal cell line you can leave these cells in culture, even if they do not grow until they reach a point when they actually start growing, however this can take a very very long time. Now if your Hayflick limit is a long way off from your starting point you could create a pool of primary cells which you can expand and then use over a set number of passages to perform your experiments. However your cells will more than likely end up loosing some of their primary characteristics as they may well have changed or accumulated mutatations or changes that make them different as they are in effect being selected for their increased growth potential. If you want to actually generate a stable and immortal cell line then you can actually transfect/infect with a telomerase expressing vector, and the ATCC has used this to generate cells with extended proliferative capacity. Another approach is to use a temperature sensitive or conditional SV40 Large T antigen expression system which allows you to make these cells immortal.
If it were me doing the experiment and I had access to a readily available source of primary tissue I would just purify the cells a Katrin has said and do my experiments in a primary culture. This has the benefit that the cells will most likely behave in a manner that is closer to their natural behaviour in vivo, and the different number of primary cultures should give you a wide spread of phenotype from a number of different donar individuals or animals. Secondly, the time it takes to generate and characterise a stable and immortal cell line can be quite long and all the transfections/infections and selection might generate a series of heterogeneous cell lines with very little resemblance to the primary tissue.
Good luck
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This is a viral disease belonging to family Circoviridea and it considered the smallest viral particle among the avian pathogens. Until now it had been difficult to find a cell line for its multiplication.
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I think Dr. Mohamed if you try tissue culture from folliculoma as CAV multiplyinMDSC but PBFD tropism is feather follicle epithelium.
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I plan to culture the HUVEC cell line CRL-1730 on a permeable membrane. Which one is better- polycarbonate membrane, polyester membrane or PET membrane? Also what is the optimum pore size for studying HUVEC permeability- 0.4, 1 or 3 micrometer?
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I'd guess that your cells would readily migrate through the 3um pore membranes...
G-361 melanoma cell line
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Has anyone worked with it? I don't know if I'm doing something wrong. It's an adherent cell line but after a week in culture cells are still in suspension. I'm using McCoy 5a medium supplemented with FCS (10%).
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Hi Maria, all the recommendations above are useful. Although I never worked with this cell line I worked and established melanoma cell lines from patients and have experience on that. 1) Do the cells release a lot of melanin to the media? If this is the case and if the cells are still in suspension I would centrifuge them and place in half fresh media (not 100% because they may condition the media with growth factors). Melanin could be toxic and indicate that the cells are more differentiated and therefore will soon die. 2) Reduce the size of the flask to increase a little cell density if it is too low and do not use big flasks (some cells doesn't like the bottom part of the flasks since the CO2 cannot easily reach the area). 3) I remember my primary and later adapted as cell line, melanoma cells like galactose added to the media. But I didn't see that these cells require that supplement. 4) Once they are attached I will use only Trypsin EDTA 0.05% (not 0.25%). Good luck!
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I am using MEM and 15% fetal bovine serum (FBS) for growth. I perform a phosphate buffered saline (PBS) wash and later add trypsin EDTA solution. When I seed the cells in 96-well plate, by the second day they are detaching.
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You can try follwoing remedies, one by one: 1. While sub-culture, you may follow following steps-a. First wash with PBS, add trypsin -ETDA for about 2 min, you may keep in CO2 incubator to maintain Temp, watch in microscope, cells may start appearing to detach, b. Add about 5-10 ml (depening on size of culture dish) complete medium (with 10 % FCS), c. flush culture with this medium gently so that all cells are in medium, d. centrifuge ~1500 rpm for about 2-3 min, e. remove supernatent and suspend cells in complete medium and plate them. You have to be careful to not leave cells for longer in trypsin-EDTA and also not to centrifuge at very high speed, otherwise cell viability drastically goes down.
2. You may try to use, DMEM, which may give better results
3. Check your FBS, it should not have kept longer at 4 C (more than 2 months) or at room temprature (more than a week), this inactivates many growth factors required for growth of cells,
4. If these things do not work, check for mycoplasm contamination using standard kit. In case, positive, better to change the culture. I hope you would be able to overcome the problem. Best wishes.
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I am trying to express some bacterial proteins in mouse macrophages (Raw 264.7), but I am unable to transfect the cells. If someone knows the protocol please let me know.
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Please make it clear your construct is okay for expression in the cell line
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I have a problem with properly counting cells in a chamber. My calculations seem to be wrong. I'm using a BHK-21 cell line to grow a virus in a 24 hour incubation. Concentrating cells in wells (96-microplates) is too high for me, but when I do the calculation for the results I need to have a higher concentration. Maybe the mistake is in the desired concentration?
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Are you sure that you are using a Burker's chamber (not a newbauer or others)?
I can suggest you to perform a count using trypan blue in order to seed only viable cells (adjusting the count on the basis of %viability)-
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I am looking for a cell line which doesn't show constitutive activation of the PI3K-Akt pathway. I am planning to do a protein interaction study using activators of this pathway like Insulin, hence I need a cell line which won't show constitutive Akt activity. Another criterion is transfection efficiency. Can someone suggest a good cell line for my intended study?
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I have been using HuH7 cells, and from what I have seen they don't show constitutive Akt activity. They respond to very low concentrations of insulin (1nM), and I have had good results transfecting them with Lipofectamine.
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The slow growth is limiting the amount of experiments we are able to do. We have tested several mediums and serums and nothing seems to help. Does it need any particular growth factor?
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It is important with Caco-2 not to let them reach confluence before subculture. If they grow to confluence they adhere very tightly, are difficult to disaggregate, and do not plate well.
Make sure you know the number of viable cells that you are plating after subculture. 2x10e4 or 5x10e4/ml should be about right. Do not rely on split level.
DMEM/F12 should be OK (we used DMEM/F10). Not sure you need 20% serum or heat inactivation. We used 10% FBS, not heat inactivated.
I agree with your other respondents -- make sure they are mycoplasma free.
It is better to grow them without antibiotics to ensure that you have no low level contaminants in there. You MUST leave out antibiotics for at least one week before you test for mycoplasma.
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I need to work on cell lines that have mutated p53 and I am confused about the difference between many cell lines (MCF7, CACO-2, Hela cell, HEPG-2).
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HeLa cells have p53-null status.
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I want to use HEK cell for stable selection, I know 293T are not suitable, and I found a batch of 293F (supposed to be cultured in suspension but that seem to grow very well as adherent cells...). Can I use these cells to select stable lines with Geneticin?
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We have generated stable cells using AD293 cells. Selecting with 1000 ug/ml G418. They are still loosely adherent so be careful changing the media etc so as not to dislodge them. We can also grow them on acid washed coverslips for IF
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I'm trying to culture the MRC-5 cell line. Im using DMEM + 10% FBS medium. After 5 days, the confluency only increased from 10% to 40% in a 60mm culture dish and there are still some cells floating. My questions are:
1. Why is this happening and how do I prevent it?
2. How long does it normally take for the number of MRC-5 cells to double?
3. What is the best protocol to detach the cells from the dish without damaging the cells?
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Hi Omar,
Having some cells floating during cell culture is normal. And if you just established the cultures from a frozen stock it is usual for the cells to take some time before entering their exponential growth rates. From my experience, the doubling time for the MRC-5 cells is around 2 days.
Some explanations for the slow growth rate of the cells are:
1) cells were initially seeded at a very low density (I used to seed them at 3000-3500cells/cm2). You can also consult many companies' sites for the different cell numbers recommended for each culture plate (http://www.invitrogen.com/etc/medialib/en/filelibrary/pdf.Par.4786.File.dat/Useful_Numbers_Y14472_Useful_Nmbrs.pdf).
2) what is the passage number of your cells? cauze if they are overgrown, it is possible that your cells are entering senescence and stop growing normally.
3) do you have any signs of contamination? you could check mainly for mycoplasma, as bacterial or fungal contaminations are quite obvious, sometimes even by naked eye.
4) you might consider supplementing your medium with non-essential amino acids, check also that it contains L-glutamine and pyruvate.
You can detach those cells with Trypsin-EDTA, following the standard protocol recommended by the ATCC (http://www.atcc.org/attachments/17396.pdf). Usually, they get off quite easily, just make sure you rinse the plate once with PBS before adding the trypsin, to remove any traces of serum. If you are afraid that trypsin might be too strong, you can even use plain EDTA at 1mM final concentration. The cells will detach but it will take longer than Trypsin-EDTA.
Good luck!
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I'm currently growing Caco-2 cells in T-Flask before transferring them to a Matrigel embedded system. The problem is that obviously these cells don't like the plastic and grow in big groups of cells that eventually cover the whole surface. I was wondering whether plating them on collagen or fibronectin coated surface could make them feel happier and grow faster. Any hints, suggestions or recommendations?
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Hi Eric,
I worked with Caco-2 cells for a while and I had no problems with cell clustering. I just used Corning T75 flask without any additional treatment. The secret is to disgreggate the clusters very well, doing "ups & down" with the micropippete before seeding them.
In the only case were I needed to treat the surface was when I used glass coverslips. In that case, poly-L-lysine treatment was enough to make these cells stick to the surface.
Good luck!
Sandra
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I received a construct to be used with the Flp-In T-Rex system but the host cell line I need for my experiments (MDCK or any cyst-competent cell lines) is not commercially available.
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This may be outdated at this point, but if you're still looking it may be worth checking here (doi:10.1152/ajprenal.90406.2008).
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Certain results that I was getting from these cell lines are not being seen anymore.
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I have not used this M12 cell line but I worked with other metastatic cell lines and it is true that some of them can evolve after 2-3 weeks (5-7 passages) in cell culture. But you can probably affect their properties after only a few passages if the conditions of cell culture are not optimal. Leaving the cells too confluent, leaving the trypsin too long or using a new medium (new serum or supplements...) may stress the cells out and affect their properties. If your conditions of cell culture are optimal, the best is to thaw a new batch of cells frozen at an early passage, expand them, freeze them. Having a big stock will permit you to always use 'fresh' cells.
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Does anyone know what ovarian cancer subtypes HEY and SKOV3 cancer cell lines are? I looked though the literature and I have found some conflicting descriptions.
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SKOV-3 was initially a serous ovarian carcinoma
How can I track a transgene that has been incorporated to a specific cell line using a FISH-based approach?
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I was having a conversation a couple of weeks ago and this matter came up. How can we track a small transgene when inserted in a specific cell line using a FISH-based approach? In my mind I am not sure that this would be possible since the transgene is just a few bp in some cases. I have read that if BACs were used the minimum size of the probe is 100-200 kb and in cases of phages the minimum size drops down to 70-100 kb. Is there another FISH-based technique to track such a small transgene? I found this publication about a new approach to produce Oligopaint FISH probes (Beliveau BJ Proc Natl Acad Sci U S A. 2012 Dec 26;109(52):21301-6. doi: 10.1073/pnas.1213818110) sized down to 10kb (at least for humans). Is there a way to go lower than this or the fluorescence intensity would not be enough to be detected? In the end, is a FISH-based technique the optimal approach to track the transgene or ultimately other methodologies are more appropriate?
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With FISH, especially for transgenes you can go down to 1.5-2 kb in size, assuming the sequence is not repeat containing. We can stably detect HPV (human papilloma virus) integration that is about 4-4.5 kb in size. Fosmid probes are very good options, yuu can fins them at UCSC web page. For the sensitivity you would need, it is recommended the use of indirect labelling in combination with a robust signal amplification. We use biotin labeled probes followed by Fluorophore conjugated streptavidin incubation, after this we apply biotin labelled anti streptavidin antibody and followed with a third layer of fluorophore conjugated streptavidin. With this we were able to show 1.5 kb probe integration. The oligo approach is good, but a similar commercial service is available from Agilent. Sure FISH, in fact that can be used for you custom probe design, too. You get rid of the trouble of repeats and have an optimal probe size and labeling. Contra, is the limited flexibility, you are bound to the probes generated by the company and so far they are not too flexible in making color variations. For really small thing, you may consider the use of padlock probes in combination with rolling circle amplification (RCA). See Nat Methods. 2004 Dec;1(3):227-32. Epub 2004 Nov 18.. It is robust tool, but it needs intensive optimization. In this way you not only could detect small sequences, but would be able to detect point mutations.
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My hela line grows very slowly.
I changed my source and got new source of hela line from another institute, but when I passage this new source, the new flasks grow slowly again.
what can I do for this problem?
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Try different % of FBS you can use 10,15,20%.
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In this webpage you can browse Ic50 for a great number of compounds in a great variety of cell lines
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I want to know if the low temperature can change the viability of cell lines.
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Not much of damage will be made to the cells. The viability ratio of the cells get affected only when the temperature goes beyond -50 degrees. The major concern with respect to your question is that the shelf life of the cells reduce when you get it back to -80. It may stay stable for maximum one year and this also depends on the handling and storage media used.