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How do you solve problems that arise from dimethyl sulfoxide (DMSO) in cells treated with DMSO diluted kinase inhibitors?
Sep 18, 2012
We are pre-incubating live cells 10-20 minutes with kinase inhibitors (stocks in DMSO) or vechicle (~0.1 - 0.5% DMSO in media) before adding agonists and see effects in control cells from the DMSO-pre incubation (e.g. drug response reduced). Has anyone experienced the same, and how did you solve it?
Normally, cell lines commonly used for screening are not sensitive to DMSO in this concentration range and you should not expect any such effects. However, this could be due to the specific cell line (or primary cells?) you are using or, more likely, low quality DMSO. I work for a chemistry-driven CRO servicing pharma industry and we routinely screen new batches of DMSO we use to prepare screening compound libraries for cytotoxicity on some common cell lines, such as HEK293. If you want to be on the safe side in this regard - buy something like Sigma-Aldrich Bioreagent grade DMSO.
Hesperidin probably is a very complex molecule to dissolve because of the simultaneous presence of a hydrophilic and a lypophilic part. As Dr. Bangdula suggested you can try to use ethanol, but the final concentration of ethanol in cell culture medium should be lower than 0.01%to avoid a direct effect of EtOH, so may be that you get a re-precipitation of the drug. In that case you may try to complex hesperidin with FBS: first make a mother solution of hesperidin in FBS, dissolving it in the appropriate amount of FBS , warming it and put in a sonicating water bath and then take the appropriate aliquots for your required concentrations. FBS is made mostly of Fetuin A, that has a tendency to surround the poorly water soluble compounds (even inorganic and complete insoluble hydroxyapatite) with a layer and bring them in solution, the compound is then readily available by the cells and the micelles obtained are very stable (I used this method when I was treating cells with fatty acids with a long chain). Definitely DMSO at any concentration is not an option,because in a drug screening that I was doing, DMSO controls had always the highest readings of all the drugs tested, regardless of the concentration, it does interfere with too many cell functions.
I accidentally put a piece of tissue in a tube containing medium+DMSO to be used for RNA extraction later on. 15 minutes after putting the tube in the -80 I realized that I made this mistake so I washed the tissue with medium and put it in an empty tube before transferring it to the -80 once again. Do you think the RNA got degraded because of the DMSO?
Hi, I started to label my dsRNA using Alexa Fluor 488 (ULS LABELLING REAGENT). Interestingly, the protocol suggests that you add DMSO to solubilize the Alexa fluor and mix with the dsRNA, incubate for 90 degree for 10 minutes and snap cool afterwards. So it seems that the DMSO prevents the secondary structure formation of the dsRNA upon denaturing it so that the fluorescein molecule can attach to it. I was planning to extend the incubation period at 90 degree for 30 minutes rather than 10 minutes to increase a more efficient binding of the fluorescein. Does
Non-essential oils are usually not soluble in DMSO and later in the medium. Try other solvent such as ethanol, use carriers such as cyclodextrins or albumins (BSA, FBS), or use biocompatible emulsifiers. You can buy expensive proprietary solubilizing agents. Do not forget to control for any of these. The oils might separate when diluting into the medium (milky clouds) or even build greasy drops. Undissolved lipids are highly undesirable. The added lipophilic compounds will be adsorbed by the plastics, shuttled around by medium constituents (e.g. albumins from BSA), and will typically be quickly absorbed by the cells.
Any advice on how long can we use the diluted natural compounds (DMSO Stock) in cell culture media?
Jun 1, 2014
Will compounds (dissolved in 100% DMSO) diluted in cell culture media lose stability at 4 degree Celsius?
The problem is not that DMSO is not stable at 4 C, the problem is that is toxic to the cells and, in the best case, make them behave erratically, interacting with a lot of cellular enzyme and receptors,making impossible to obtain reliable results. So that is why when people thaw cells froze in 10% DMSO,they change the media after the cells are attached. If you have to, the way is to dissolve the compounds in ethanol and then add to the media with a final concentration of no more than 0.01%, because otherwise even ethanol will interact with the cells falsifying your results.
What is the freshness of DMSO and methanol solutions?
May 19, 2014
I would like to ask you, how long a flavonoid solution in DMSO and methanol solvents can be kept in the fridge? Is there any shelf life to use it for studies?
The best test is to measure the uv-vis-spectra of the prepared solutions. If the absorption intensity and position of the known absorption bands are unchanged, the solutions can be used for further study. Otherwise, it is recommended to prepare fresh solutions.
Quite honestly I'm baffled that so many researchers believe DMSO is toxic. Usually, if there is observed toxicity from DMSO it means the DMSO is impure since it can carry toxic substances through biological membranes so efficiently.
Well, cannabinoids -and specially endocannabinoids- are really delicate molecules, they tend to oxidize and they are susceptible of degradation by different enzymes. It would be nice to know if the cell line/type that you are using express cannabinoids degrading enzymes, if it's the case, I guess that AEA or 2AG are going to be unstable in your culture. We found a degradation effect of 2AG (60uM) within minutes after application, and this was prevented by coincubation with an inhibitor (JZL 184 1uM) of its degrading enzyme, MAGL (http://www.ncbi.nlm.nih.gov/pubmed/24533119, supplementary figure 3). If your cells express this kind of degrading enzymes, then you should inhibit them before cannabinoids application. However you can find several examples of long-lasting treatments with cannabinoids in culture medium.
I synthesized PAMAM-OH dendrimers by using DMSO solvent. Now I find difficult to remove my product from DMSO. Is there any methods to do it? I have tried distillation but didn't work. The product is thermal sensitive and a temperature above 50 oC may destroy it.
I assume your PAMAM-OH is water soluble, if so I can offer to use membrane ultrafiltration. Depending on the Mw of PAMAM-OH, 0,5KD or 1 KD membrane which are resist to DMSO, can be solution for the necessary purification. you can follow
first dilute the reaction mixture with deionized water to 10-20 mL, then transfer it to 200 mL LPR cell which has alreadt installed DMSO resistive dialysis filter, after that apply 1-2 bar pressure for 8 hours finally check your filtrate and retentate. The retentate shoul be pure eneough for your desired application.
I recently received my order of ACS grade DMSO and it came in a semi transparent plastic bottle. This is the first time I have come across such packaging. Previously, I have only used DMSO packed in dark glass bottles. Will this affect the quality of my DMSO?
DMSO has got hygroscopic nature and is highly light sensitive. So it must be kept in dark container and in moisture free places. I would prefer to aliquot it in small vials (for e.g., 1 ml in each eppendorf) wrap with alluminium foils and freeze them at -70/-80 degree celsius. Take out one small alliquot each time (when required), thaw it and use it.
I want to carry out the MTT Test, but in the literature, there is no standard test. The methodologies vary a lot from one another. Some people talk about shaking before optical reading, some don't; some care about the DMSO quantity added, some don't.
Does this mean that I have to set a specific methodology for my purpose?
Apr 24, 2014
From my personal experience I would say that shacking before reading is not essential but improves homogeneity within each sample. For DMSO quantity, refer to the manufacturer's datasheet and try to follow their procedure guidelines if they provide them. Generally, the main reason for unsatisfactory results is inaccuracy in initial cell seeding and in pipetting the MTT solution (always add the same volume of solution in each well, taken from the same stock solution).
MTT assay is quite an easy and straightforward assay, you will soon become confident with this methodology. Good luck!
The story of DMSO always fascinates my scientific quest. The more I search about this ‘persecuted drug’, the more it captivates my attention. I heard that there was lot of controversy about DMSO in late 60s and 70s. Is it an example for the ‘dark side of human nature’? Could someone from that age comment on this? Or what is your view on the whole story?
It dissolved properly in 1N HCl but for enzymatic assay I require pH 7.5 in tris-HCl buffer. At pH 7.5, the solution starts precipitating. I also tried to dissolve in DMSO and Acetone but in both solvent, it was not dissolved properly. It would be of great help if you suggest something to make it soluble at pH 7.5.
I have dissolved a hydrophobic compound in DMSO (final conc. 100mM) for my cell culture experiments. But when I tried to dilute the stock with PBS or media the compound precipitates. Later, I diluted the original stock with DMSO to 10mM. With this stock no precipitates appear when added to the medium. The problem is that the DMSO (>0.2%) has shown to induce effects on my experimental cell line. The amount of DMSO equivalent to 10mM stock will be more than 0.8% in my control experiments. Is there an alternative way of diluting the 100mM stock solution to avoid the precipitate formation?
I had the same problem with DMSO. When I tried to dilute my 100 mM DTNP stock solution in Ringer's solution to get the 100 µM working concentration I had to vortex it for some time to remove the shade and get a clear solution.
DMSO inhibits Methionine-Sulfoxide reductases in concentrations >1%, is cyto- and genotoxic. So you should be aware of having a final concentration of DMSO as less as possible.
How can I make a polymer inclusion membrane using DMSO as a solvent?
Apr 2, 2014
I have synthesized an organic compound having solubility only in DMSO but because of the high boiling point of DMSO it is not getting vaporized thus I am not able to make a membrane. And if i take a mixture of the solvents : dicloromethane and DMSO then phase separation occurs.
As correctly mentioned by Manoj, DMSO is aprotic. In fact, the pH concept does not apply to such solvents, because it is applicable only for H2O; hence, we know of super-acids like H[PF6] and such. Thus, you cannot talk about a pH of a pure anhydrous DMSO whatsoever. Compounds dissolved in pure DMSO will have wildly different pKa values than when dissolved in H2O, thus if you measure pH, which based on water, the measurements may be very different than expected. Literature is abundant on this topic.
I need to dissolve my propolis extract (concentrated--> evaporador rotatorio) in DMSO not exceeding 0.01% in potato dextrose agar medium (200ml) and final concentration of propolis 400ug/ml in agar medium.
If this is a polar extract, like in ethanol, methanol, ethyl acetate or water, just directly dissolve in luke warm agar media- best to avoid DMSO, but if it's a hexane, chloroform like non-polar extract, then make a higher stock in 1% DMSO, or say propylene/ ethylene glycol or even some Tween 20/ Triton X (~0.1% or less) like detergent, then you can reach that final concentration. Make sure to use the same detergent, DMSO, or glycols in control plates without the propolis extract to make sure that the effect/ inhibition is not from the dissolving-agent but from the extract !
I am carrying out an assay in which I am using an mCherry (RedFP) expression vector as a measure of transfection efficiency. However when cells are treated with XL 413 and KU - 55993 transfection efficiency appears to be reduced by ~50% when compared to DMSO treated samples.
It is not clear if this effect of drugs is specific to mCherry or all transfections are affected similarly. This is important as my second readout from this is assay is GFP and these values are normalised for the transfection efficiency seen in mCherry transfected samples. Flow cytometry is used for analysis.
You have drug X and dissolve it in DMSO at a concentration of 100 mg / mL. Your goal is to create an aliquot of this drug with a final concentration of 200ug/mL. The solvent you will use to dilute it is cell culture media. Is it possible to create this aliquot while keeping DMSO below 0.1% v/v? I have tried this and could not get it below 0.2%. If anyone can please help and show how they arrived at the answer I would really appreciate it!
Could anyone explain this? A lot of papers ( as "Resveratrol attenuates brain damage in a rat model of focal cerebral ischemia via up-regulation of hippocampal Bcl-2" e.g.) told us that they treat rats with 30 mg/kg i.p Resveratrol dissolved in 2% DMSO saline.
If we do the maths:
Resveratrol solubility in DMSO =16mg/mL ( A. Amri et Al. 2012)
Average rat weight = 300 g
Resveratrol treatment for 300 g at 30 mg/kg = 9 mg for 300 g
9mg of Resveratrol needs 562,5 microlitres to be effectively dissolved.
For 562,5 microL of DMSO we need 28,125 milliliters of solution (DMSO 2% saline). They injected 28 milliliters of This into each rat once a day? What am I missing here? Am I a complete disaster with maths or there is something strange here?
I suspect that your solubility information is not correct. Cayman Chemical states that reservatrol can be diluted in DMSO and other organic solvents at 65 mg/ml. That gives 9 mg/65 mg/ml=0.138 ml of the straight DMSO stock. That still gives a dose of 6.9 ml twice a day. It may be that the solubility can go higher, or it can also be that they are dosing with a suspension. I have had in the past to gavage rats with a suspension when solubility was not in my favor.
Many companies will print solubility data that they have, but they do not necessarily attempt to find the absolute max that you can dissolve a given chemical in a given solvent.
I’m designing a set primer (with melting temp about 60°c) for PCR of a genomic fragment (5 kb). In this region of genomic DNA, there are several very stable secondary structures (about ΔG = -190 kcal/mol at 72°c) that are distributed in 5 kb. Would DMSO addition to this PCR, solve this problem? What concentration of DMSO is needed for this PCR?
So the serynge is filled with a ligand A solution and I will assume that the cell is filled with a ligand B solution (so it`s probably a protein-ligand experiment).
So i will continue with my previous opinion. The endothermic values you see in your thermogram is the interaction beetween your protein and your ligand. The exothermic values you see in your data and graph is probably the dilution of your protein solution.
I would recommend you perform a simple test in your ITC, filling the serynge with protein solution and maintaining cell filled just with your buffer. If I`m correct, you will see just exothermic peaks in your thermogram, showing the dilution heat of your protein.
So you can subtract the heat of your diluting experiment of your thermogram or, if you desire, just normalize your experimental data subtracting the final exothermic vales of your data (the constant exothermic value you see in your graph).
I used a Heme Oxygenase-1 enzyme inducer, named Cobalt protoporphyrin, and dissolved in DMSO and stored the aliquots in -20 degree. I would like to know the maximum time that I could still use it giving similar results. If anyone has previous experience, please let me know?
I'm a grad student in biochemistry and now trying to grow NK92-MI cells from ATCC but they all die by day 3. Cells were frozen in 90%FBS/10%DMSO at a cell density of 2x10-6, thawed and seeded at 2.5x10-5 cells/ml following ATCC protocol. I'm using alfa MEM supplemented with L-glutamine, 2-mercaptoetanol, folic acid, inositol, 12.5% HS and 12.5% FBS.
I have some questions...
1. Should I feed at day 2 or rather let them settle after thawing and feed at day 3?
2. I'm spinning down to remove all DMSO after thawing and resuspending with fresh media to seed. Should I not spin down?
3. Is there any other media they grow better with?
I don't have specific experience with dissolving alginate, but a lot with insoluble new pharmaceuticals. If water, ethanol and DMSO don't work, acetone is sometimes a good option. Acetone mixes quite well with water for testing your molecule in biological systems and is not too toxic in cell culture when dissolved up to several percents in cell culture media.
With kind regards,
I want to find out the reduction potential of DMSO solvent. I tried with cyclic voltmetery but it showed no results. So if anyone knows about the reduction potential please share with me with its reference or if someone tell me what I can do to find it?
yes， i focus on non-aqueous li-air battery using DEMS.
Has anyone observed an increase in fluorescence signal over time at higher concentrations of fluorescent reagent?
Jul 22, 2014
I am trying to measure fluorescence polarization using a TAMRA-based ubiquitin reagent. The reagent is dissolved in DMSO and the diluted into a fluorescence polarization buffer containing salts, BGG and CHAPS. Then an enzyme was added and the change in polarization ratio was measured over time (measurement every minute for 90 minutes).
I made different concentrations of Ub-TAMRA reagent by diluting it in the buffer. On measuring the total fluorescence, I observed an initial drop (probably due to photo-bleaching), followed by an increase in the total fluorescence (the polarization value also had a bump). Then the fluorescence intensity stabilized. This happend at a particular time point during the assay. The "event" happens at earlier time point for diluted samples and the intensity of this bump is lower at lower concentrations.
Has anyone observed similar effect at higher concentrations of the fluorescent reagent? I am using a Pherastar for measurement.
The emission intensity of a dye can increase at high concentration for a couple of reasons:
1. If the emission and absorption spectra of the dye has a very good overlap, they can undergo intermolecular fluorescence resonance energy transfer (FRET); that is the emission of one dye molecule can act as an excitation source of another dye molecule close to it. If the concentration is too high, the dye molecules can be pretty close to each other to meet the requirement of FRET distance (energy donor and acceptor should be within 1-10 nm distance for FRET to occur). If you are seeing the increase over time, then there is a possibility that over time, the dyes are aggregating (may be your enzyme is inducing dye aggregation or dyes are self-aggregating), making the dyes close to each other for FRET to occur. I don't exactly know the structure of your dye so that I could check the excitation and emission spectra. I just found information about 5-TAMRA which has abs maxima at 541 nm and emission maxima at 568 nm.
So there is a great possibility of spectral overlap and inter-dye FRET for the dye.
2. Some dyes (e.g. SYBR Gold) show higher fluorescence when they bind to or intercalate into any molecule (e.g. DNA or others). It's worthy of looking if there is any possibility of happening this kind of phenomena in your system since you have enzyme added to your dye solution.
Hope that helps!
I dissolved the drug in DMSO and its completely dissolved but when I diluted in cell culture media formed crystals. I used (2-hydroxypropyl)-beta-cyclodextrin for dissolving the same small molecule for VIVO study. Can I use (2-hydroxypropyl)-beta-cyclodextrin as a solvent for Vitro study also? if I can, up to which concentration is possible to use it?
I am doing an anti-mitotic study and I am planning to use DMSO to dissolve my plant extract in order to make a test solution. However, some articles would state the concentration of DMSO should be 0.1% while others used more than that. What should be the proper concentration of DMSO to be used in a sense that it would be non-toxic to sea urchin cells? Can someone please provide or attach an article, a journal or a link that would state the proper use of DMSO in terms of its concentration when used as a solvent. Thanks!
You would want to perform solubility tests for your compound in different temperatures of DMSO with or without stirring. Some substances dissolve on their own when left overnight. 25% DMSO is not advisable. Keep it below 0.5% if you can because DMSO is known to be genotoxic also.
I have to use 3-MA in my cell culture at high concentration. II know that 3-MA is not very soluble in water, so the solution would be solubilize 3-MA in DMSO, but I know that DMSO generally alters the phenotype of cells in culture.
hello! I am using DMSO as a solvent for a compound for in-vivo trials in adult mice. I am delivering it IP. Does anyone know the maximum tolerated dose in this situation? I have come across varying answers from 1%-20%! Thank you.
Dear Teresa, lesser the DMSO used the better, reason being the toxicity associated with it. Try dissolving the drug with the DMSO concentration less than 2-5%. However, if needed, you may have to go higher.
As recently shown DMSO enhances electrospray response (see Hahne et al, Nat Methods. 2013: DMSO enhances electrospray response, boosting sensitivity of proteomic experiments. http://www.nature.com/nmeth/journal/v10/n10/full/nmeth.2610.html). We also have seen higher identification rates in complex samples using 5% DMSO in the LC-buffer for our Orbitrap Velos Pro: 30% more PSMs and 20% more peptides and protein groups.
What we like to know is your experience with DMSO effects on the hardware side (MS and/or LC). It is pretty clear that there is an enhancement to IDs but before going to high-throughput with DMSO in the running buffer we like to ensure that there is no negative impact on the hardware. Some engineers recommend avoiding DMSO due to its effect on the ion optics pathway of the MS.
Bernhard Kuster gave a talk at the recent ASMS meeting and presented his findings. In summary it was that he does need to clean his instruments more frequently, but they are persisting with DMSO as the benefits are tangible. Don't quote me, but I think the time between cleaning was halved, so every 6-7 weeks instead of every 12-14 weeks.
He also included feedback from others that had tried DMSO. Most did find an improvement, some found little difference, and some even saw a decrease in identifications. Clearly you are seeing an improvement.
We haven't started using it yet as I am concerned about the cleaning aspect. I guess you need to decide for yourself if the improvement is worth the more frequent cleaning. Kuster certainly came to the conclusion that it is worth it.
Even at 0.1% of DMSO could be defined that it slightly decreases cellular functions according to some authors, and this was demonstrated that it gets significant at 0.5%. Use as low as possible DMSO is the best practice and you should always have a solvent control. But I must still add that type of the cell you are using would always make difference for sure. Good luck...
I am trying to do an assay in which I will be making serial dilutions of a drug dissolved in DMSO using a buffer solution. By doing this, the concentration of DMSO in each dilution decreases. But I want to maintain a constant concentration of DMSO and see that only the amount of drag varies in every dilution. How should I do it?
The protocols I have read seem straightforward on differentiating HL-60 cells, however I am not having luck using 1.3% DMSO and/or 1uM ATRA (all trans retinoic acid). I culture in 1640 RPMI + glutamine and 25mM HEPES, 5% FBS, and Anti/Anti. The cells are growing nicely (doubling time 24Hr). The cells are seeded @ 1X10^5 cells at time of treatment. However, they continue to grow after exposure to the differentiation medium. Passage is low ~ P12 from ATCC vial. I tried testing different cell densities with no luck. Any help would be greatly appreciated.
Your HL-60 cell line may not be what you think it is anymore depending on the source and who started the line and how it has been thawed/cultured initially. Keep in mind that the cells are cryopreserved in 10% DMSO, which also induces them towards the neutrophilic phenotype. If the DMSO is not completely removed when the cryopreserved cells are thawed, they are being induced to differentiate, proliferation slows down and the cells you eventually end up with after weeks of culture, will be unreponsive to DMSO! Suggest you get new HL-60 cells from a reliable source, follow a protocol which removes the DMSO as much as possible on the first days of culture, and then try again. I am attaching our protocol for doing this. It works every time! Good luck. Feel free to share this little know issue with others.
I have aqueous and ethanolic extracts which I plan to reconstitute in PBS and DMSO before doing enzyme inhibition assays on alpha amylase and alpha glucosidase. I will need to do serial dilutions to determine the minimum inhibitory concentration of each extract, but what should I use as the starting concentration of the crude extract?
This is a very broad-based question! I think a good approach would be to choose a positive control. Look for a known (commercial) inhbitor(s) of these enzymes and use the reported Minimum Inhibition concentration of the known inhibitor as the starting point (concentration) for your extract. Based on the preliminary result you can use increasing concentrations of the extract (if the extract is less inhibitory than the positive control) or serial dilution of the extract (if the extract is more inhibitory than the positive control).
You can make a comparative study between the positive control and your extract to get an ideal how good the extract is in enzyme inhibition
I want to determine pancreatic lipase inhibition activity with 4-methylumbelliferyl oleate as a substrate. I have checked a number of published manuscripts where they dissolved 4-methylumbelliferyl oleate in 13 mM Tris-HCl buffer (pH 8.0) or Mcilvaine’s buffer (pH 7.4). However, 4-methylumbelliferyl oleate is not soluble in water. It is only soluble in 2-Ethoxy methanol or DMSO. If any of you did this assay before, please let me know how you dissolved 4-methylumbelliferyl oleate, and if I dissolved in DMSO is it ok for the assay?
Feb 1, 2015
You need to dissolve the 4MU-oleate in a minimum of DMSO and then add it to the assay solution.
I want to know if there is any way to increase the evaporation rate of a DMSO-PAN solution without external applied energy (vacuum, temperature, etc). Would a surfactant or salt of some sort be able to achieve this?
I am a student of MS, and have started work on HIT- T15 Cells. They were cryopreserved in 10% DMSO containing media. ATCC recommends Ham's F-12 media for its growth, however researchers are using RPMI-1640. I also tried to revive it in RPMI media supplemented with pen-strep, Na- pyruvate, 50uM 2 Mercaptoethanol, 10mM HEPES PH7.4, 1mM Glutamine, but it is not growing in TPP Culture dishes. TPP culture dishes are cell culture treated but not coated. I am working on C2C12 Cells, and they grow well on TPP dishes. Do the HIT-T15 cells need a coating material for growth? I cannot find any relevant information about HIT-T15 cells. I just read one paper in which they reported that DMSO is not suitable for encapsulated HIT-T15 cells, but as I don't use encapsulated cells, this has nothing to do with it.
To answer your question to Tsehay regarding glucose concentration in HIT-T15 cells when thawing: We sometimes do long term cultures at 0.8 mM glucose. If we freeze these cells back and then wish to wake them up again for more experiments, we must wake them up in 11.1 mM glucose for a few days or up to a week before putting them back into 0.8 mM. If you wake them up in low glucose, you will lose a lot of cells. I do not believe that we explicitly mentioned this in our papers, but if a new person in the lab forgets or was not told, it is apparent when the low glucose cells are thawed out and don't survive well. We have an older paper about the effects of various culture conditions on these cells - JCI 90:320-325, 1992.
RPMI 1640 is 2 g/L glucose, which is equivalent to 11.1 mM. I would not add more than 2 g/L glucose to the culture media unless doing so is part of your research design, the cells don't need it and lose the ability to secrete insulin when maintained at 11.1 mM.
Which solvent do you choose to prepare DPPH solution: methanol or DMSO? Are there any significant differences between these solvents?
Mar 11, 2014
So far, I've used DPPH solution in DMSO because my samples of naringenin derivatives are only and only soluble in DMSO solvent. The maximum absorbance was determined at 523 nm. Reviewing the literature I've noticed that some people used samples dissolved in DMSO and DPPH dissolved in methanol. Is it good that I use DMSO solvent to prepare DPPH solution? Do you know if DMSO has influence on radical scavenging effect or antioxidant activity? Please write.
Because your samples (naringenin derivatives) are soluble in DMSO and you want to evaluate the DPPH scavenging of the same, so it is preferred to use DMSO as solvent to dissolve DPPH if possible. But you should dissolve your standard or reference compound in same (DMSO) which is more necessary to get exact comparative view.
In my experience, several antimicrobial compounds are not soluble in water and therefore you need to use various solvents such as DMSO, DMF, ethanol...
When doing this, the idea is to dissolve the compound in a very concentrated form, like 100x the working concentration. You can then use this ultra concentrated, yet dissolved stock to generate your testing dilutions. In this case, the dilutions can be made ( using either water, growth media, etc). This will give you a good range of compound concentrations while limiting the exposure to solvents needed to dissolve in the first place.
If you give a few more details about how you are setting up your dilutions, what the drug is, perhaps others will have more information to add.
Some researchers suggest equilibrating cells on ice when adding cryosolution with DMSO, while others on the contrary recommend equilibration at room temperature to facilitate transport of DMSO into cells. What are your cryo experiences with eg. clinical doses of MSCs. Importantly, does anyone know a systematic head-to-head comparison reference to be recommended?
MSCs are generally regarded as rather easy cells to cryopreserve and I would agree based upon my own experience with the earlier answers that adding the DMSO on ice is fine. DMSO permeates cells in seconds so 10 minutes of equilibration should be more than enough. That said I would suggest that you compare protocols from the literature in your own lab to be sure that an unidentified variable is not impacting outcome. Simple things such as the solution into which you put the DMSO as well as cooling rate, whether or not you use nucleation to initiate freezing and storage temperatures can impact cell yield and viability.
Have you ran out your extracted DNA on a gel? You want to have high molecular weight genomic DNA for RAD lab work. Essentially you're using 1 or 2 enzymes to cut up the genome, so if your DNA is degraded then many of your cut sites may be broken apart, and thus the enzyme won't recognize them. I could also imagine that if you're DNA is highly degraded then you could potentially end up with many fragments being smaller than your desired size range. So, even though the adapter would stick to them, they would be thrown out during size selection. I would suggest running out some DNA extractions on a gel to see if there's any high m.w. DNA and use a fluoremeter to check DNA quantity. If there is some high m.w. DNA, then digest some of that sample with your enzyme(s) and see if the resulting fragments are in your desired size range. Perhaps you could find someone in your dept who would be willing to let you throw a sample or two on their lane so you could just see what you get!
Could I ask which kind of amyloid do you use and how do you prepare your amyloid to be aggregated? I bought amyloid 1-42 and want to use it to build Alzheimer disease model. I don not know how to prepare it and make it have toxic to cell. Thanks....
What Catherine, said is correct. As she said, a lot of compounds are not soluble directly in water and that is why you do your standard solutions in DMSO. Then you dilute this solution with water, medium or buffer, normally to have a final concentration of DMSO of 0.5-0.1%, at least that is how we do it. I just wanted to add, if you are working with metal complexes, I would recomend you to work with DMF instead of DMSO. This, because DMSO coordinates with a lot of metal complexes and giving you a wrong IC50 of MIC values, while with DMF you don't have this problem.
I am not sure what vehicle should be good for ATRA IP injections? First it is dissolved in DMSO or EtOH. The problem is that the stock in DMSO makes precipitate with PBS or saline upon dilution. On the other hand, solubility is much less in EtOH in 10 mM, so we have to inject 100 microlitre of 100% EtOH. We don't want to use both 100 microlitre DMSO or EtOH. Can somebody suggest me a better option?
Try dissolving directly in PBS or normal saline that has been pre-warmed to between 40-50oC with vigorous shaking. If it works (dissolves), then allow it cool down to about room temperature before injecting
I was planning to treat my cultured cell lines with c-myc inhibitor (10058-F4). The stock is 200mM in DMSO and is thoroughly dissolved---it is a clear yellow liquid. However, when I added the stock to cell medium, the inhibitor resolidifies and forms yellow powder that adheres to the wall of plate. My final concentration of treatment should be 50uM to 100uM. But in this case, when the inhibitor is no longer dissolved in the medium, I am afraid the actual concentration is much lower and the inhibitor won't work.
Does anyone have similar experience with drug treatment and could provide a solution for me?
Oct 3, 2013
u should use media as a solvent to make stock or diluted DMSO with media or distill water as a solvent to make stock but in my opinion media is better for solvent because water is also the solvent of C-MYC inhibitor in sigma.
It depends on how you are going to use your estradiol and for what purpose. As long as estradiol is able to form hydrogen bonds with your solvent, you should be able to use it but keep in mind that the solvent will stay in your prep.
It was suggested to me to include an extra negative control for my experiment - something like DMSO, but which does not affect the distribution of nucleolar proteins (as this is what my experiment tests).
I thought of DMF, but a paper shows that it may potentially downregulate a target protein for which I am testing (B23). Does anyone have any suggestions?
What is the reason for this extra control? If we used substances which had been dissolved in DMSO we always used a control which had only DMSO (in the same end concentration) in it. This sample was compared to the untreated cells to find out, if there is an effect of it.
I don't see the point of adding an extra substance, which is otherwise not used in the experiment. Even if it had an effect, it will tell nothing about the reaction to DMSO.
It also depends on the purpose of freezing. If you want to get viable animals after thawing, currently because of complexity, variability in tissue response to and permeability of cryoprotectants and water, as well as issues related to heat transfer, only relatively small animals can be frozen. Although large organs (sheep ovaries, pig and mice livers) were cryopreserved successfully, the complexity of such organs is far less than a whole animals. If, on the other hand, the purpose is to preserve the genetic material, large animals can also be frozen and viable offspring can then be generated using nuclear transfer. This was first reported in 2008 by Wakayama et al. (in PNAS 105(45): 17318-17322) when they cloned mice that were kept at -20 degrees C without any cryoprotectants for 16 years.
I've been reading through many papers and methods for solubilizing the MTT crystals in my wells, I have tried both using DMSO and isopropyl alcohol (acidified 40mM HCl) and I've noticed I get a higher abs and a better purple color with the alcohol. Using DMSO I get a more redish ting with a lower abs. What I like about the alcohol is that there seems to be consistency within papers using a wavelength of 570nm, but when individuals use dmso, there seems to be everything from 490-590 and anything in between.
Does anyone have a reason to why they choose one over the other?
Oct 17, 2013
I have used isopropanol, DMSO, and SDS in the past and have settled for DMSO not because of high OD values but because it solubilizes formazan faster for the MTT assays. There is no much problem using a regular wavelength of about 540 and 550nm and being consistent with that. The essence of the assay is too compare viability of cells (or indirectly, cytotoxicity) and this can well compared at high and low O.D values with appropriate negative control wells. In this assay, the different wells in the plate are given the equal treatment and compared as percentage viability relative to the control and that is the most important thing. Remember that there are no absolute OD values for these assays even with the same treatment, same culture media, same cells, same conditions, same solubilising agents etc. The OD values will vary from experiment to experiment and comparing different wells to your in-plate control for each assay is key to eliminating such variations. All the same thanks for drawing our attention to the higher ODs obtained with isopropanol.
I am trying to see the effect of a pharmacological inhibitor that is soluble in DMSO. Would you please advise me on the maximum DMSO concentration for IP injection into a mouse (200-500 micro lt total volume) that would not be toxic?
I worked years ago with a Biacore 3000, but didnt have to use solvent correction at that time. I would suggest asking your local Biacore Application Specialist if there is this functionality in the BIAevaluation Software 4.1 (I am quiet sure it is). If not you can use SCRUBBER (http://www.biologic.com.au/). If you have further questions regarding the DMSO correction itself, feel free to contact me.
Dissolving flavonoids in DMSO: I would like to dissolve powder apigenin for treatment of a cancer cell line. On the vendors website (sigma aldrich) it says that apigenin is soluble in 27mg/ml DMSO. The molecular weight of apigenin is 270.24
In addition to Patirck's suggestion, it important to have DMSO control in your treatment groups. Add the same amount of undiluted DMSO as you add your diluted compound for the control. For ex: if you are adding 20 µL, then add the same amount of undiluted DMSO for your DMSO control
There is no established way of giving an absolute concentration of DMSO that is toxic for every cell type.
What you should do, ideally, is performing a dose-response curve by testing different concentrations of DMSO on your cells of choice. Evaluate basic parameters like rate of cell growth and viability, and also parameters more consistent with your target experimental outcome.
That's the only way you can learn whether DMSO is toxic for your cells specifically, and the only way you can identify what is its highest tolerated concentration in the biological system and experimental setup your using.
In previous articles it says that compounds dissolved in DMSO should not exceed 0.1%. This means that the DMSO concentration in final medium (aqueous) and culture required should not exceed 0.1%, or the quantity that I will use to dissolve the chemical substance before addition in medium should not be exceed 0.1 %. This means that the final concentration of DMSO in medium should not exceed 0.1 % or the concentration I will use to dissolve my drug should be 0.1 %. Bearing in mind I have DMSO solution with concentration 99%?
My aim is to test antibacterial, anti fungal properties of some chemical compounds by the disc plate method. I have dissolved all the samples in DMSO to make the final concentrations 50 micro grams & 100 micro grams per disc, simultaneously I have tried to dissolve Streptomycin as a standard to make the final disc concentrations 50 micro grams and 100 micro grams. The problem is that DMSO did not evaporate, Streptomycin did not dissolve in DMSO.
Keep in mind that for a certain antimicrobial there is a solvent and there is a diluent that are recommended/acceptable. Some compounds are just not soluble in water or any other buffer, such as phosphate. For those compounds, it is acceptable you dissolve them in DMSO, acetone, alcohol... but, you will need to dilute them with water/buffer before you test them, meaning that even the stock solution should be a mix of solvent:diluent. Remember that any solvent that you use is likely to interact with the bacteria, fungal or any component of the broth you are using for your testing. Some compounds when pure may even interact with the disc. Just make sure that any inhibition you see is due to your compound and not what you dissolve it in, so run appropriate controls.
I have obtained a few compounds from bacteria which could only be dissolved in DMSO. Since DMSO has a high boiling point, I am finding it difficult to crystallize or solidify the compounds for further analysis. It would be helpful if there were some simple methodologies to completely evaporate or to separate the compounds from DMSO, e.g. partitioning using solvents.
I don't know what you mean by "only soluble in DMSO" but I think that your compounds are probably able to dissolve in organic solvents. For example, if the compounds are soluble in chloroform, you can remove DMSO by water extraction (3 washes with water for example) then you can remove chloroform by evaporation (a stream of nitrogen for example) and finally sublimate the traces of DMSO with freeze-drying. Then the compound can be again dissolved in chloroform for further use.
Can anyone give me a protocol to validate the DMSO used in cryopreservation of stem cells using flow cytometry?
Just speed-vac the DMSO away. Shouldn't take more than an hour.
Does DMSO have to stay completely anhydrous when stored prior to preparing cell freezing solution?
Oct 14, 2013
I have always been told in my chemistry lab that DMSO should be stored in a very dry environment, should be aspirated with a syringe through a septum, etc. to minimize exposure to humidity.
I am wondering whether these considerations apply when preparing a freezing solution for cells (90% FBS, 10% DMSO): in my biology lab, they really don't care and buy the cheapest DMSO without septum, and keep it on a shelf.
Is that bad? Why or why not?
What do you think?
I recently read the paper from Hahne in nature methods about the improvements in LC-MS performance by adding up to 5% DMSO to the LC-buffers (with quite impressing results). Has anyone experiences with this approach, especially whether it leads to contamination of the LC system or the ion source/ion stack? We have an LTQ-Orbitrap XL-ETD system and an Agilent 1200.
I wrote the first paper mentioned in the above comment. Thank you for your interest in my work. You will need to slightly modify the elution gradient. Also make sure to use a very high source temperature since DMSO has a high boiling point.
We have immobilized a protein in a CM chip and flew small molecules with different concentration on it to know the kinetic result. I am having trouble analyzing the data,since I do not know how to take DMSO solvent correction into account for the small molecules. Can anyone help me?
Hi, I figure that your instrument does'nt have the function module for solvent calibration? eg. Biacore 3000? You can use Biacore T200 instrument, on which you can include the solvent correction process in your experiment, using the calibration solutions 1-8 in the attached file. So, in the result file , you can click evaluation->add solvent correction , to apply the solvent correction to your sample curves. Then you can choose the corrected curves to analyze.
Crosslinking may affect the structure of your antibody and reduce its ability to capture its specific antigen. This effect is especially prominent when using a monoclonal antibody. I would suggest trying a polyclonal antibody, or combine 2 or more monoclonals.
If the antibody you're using is already polyclonal I would simply increase the starting amount of whole cell lysate if possible!
Make sure you also check that the antibody was succesfully crosslinked by saving the flow-through after incubation of antibody + beads, after incubation with DSS, and also by boiling the beads that are supposed to have antibody attached. You can run these saved samples and probe with secondary antibody to check.
I'm doing research about tyrosinase inhibition by plant extract but I am rather confused about what DMSO concentration I shall use. In some journals they use concentration of 50%, but there are also journals that use concentration of 1.25%. I really don't know why they use different concentration. Can somebody help me to explain the reason? Thank you.
50% is way overboard, and must be a typo or a concentration of a stock, but not the final concentration. DMSO, like any organic solvent, will significantly affect any biomolecules conformation. In addition, DMSO is involved in the oxidation of some compounds, such as tryptophan.
When performing experiments, you always need a carrier (solvent) control anyway. So just make sure you can compare DMSO to a non-DMSO reaction (e.g. compare tyrosinase activity with no inhibitor or DMSO to activity with DMSO but no inhibitor).
Ag/AgCl electrode is very easy to build in lab. In case of measuring potentials in nonwater electrolytes you can use salt bridge system with internal electrolyte dioxanemixture with water in ratio up to 75:25 and electrolyte will behavior as water solution electrolyte (I am think that it can be interesting to try, but I am not guarantee result), But of course you can use pseudo reference electrodes Pt, Pg, Au and also Ag
I want to treat my PC12 cell line with one drug, but the drug is insoluble in water and DMSO. I dissolved the drug in DMSO and media, but crystal was formed. What solvent do you suggest? What ratio should I use?
I work with Olibanum. I prepared aqueous and alcohol extraction of Olibanum and then I dried these extractions according to protocol. I don't buy Olibanum from any company (this is a traditional material) and I don't have any data sheet of this drug. I want to prepare a stock solution (for example 100X) for these extractions and then used them for cell line treatment, but I can't dissolve them in DMSO.
I have problem with the drug solvent in my work on anti-inflammatory effects of plants extracts on carragenane induced oedema (acute inflammation) and collagen induced arthritis (chronic one). Actually I am treating animals orally with a suspension of the oily extract (and indomethacin as control) in gum acacia (2% in water). But I have concerns about intestinal absorption (I need your opinion here).
The next step is to compare the amelioration obtained by the extract treatment with its individual components, some of these are in crystalline form (ex: thymoquinone: 1mm crystals) and homogeneous suspension could not be obtained. They must be dissolved to unsure the adequate dose administration to animals. Which solvent is the safest (chronic treatment), non-interfering (neither pro nor anti-inflammatory) and what are the adequate doses: DMSO, ethanol….?
I want to treat my PC12 cell line with one drug, but the drug is insoluble in water and DMSO. I dissolved the drug in DMSO and media, but crystal was formed. What solvent do you suggest? What ratio should I use?
Are you going to preserve the UCB cell OR tissue OR blood? If cell, an incubation period is not essential. A brief period should be enough (which is achieved during CPA adding, mixing, and transfer process itself)
Testing against cancer cells, I couldn't seem to dissolve my sample, even in DMSO. A suspension was formed. My preparation method: initially 1ug sample was dissolved in 10ul DMSO and further mixed with 990ul PBS to make up 1ml of total working sample, 1%.
Jan 16, 2014
what is the compound you are trying to dissolve. I can help you if you tell me in detail
Different concepts revolve about DMSO usage. Studies denotes use of 0.1% as non-toxic, contemporarily there exist reports that it is toxic, while using it as vehicle (as dissolving medium) for drug deliver. Actually should it be analyzed in a context dependent manner or how?
No, I haven't. I looked it up online because I was planning to filter a solution in DMSO, but it turned out that I didn't need it. Here is a reference:
"Consumables and accessories made up of polypropylene, polymethylpentene, nylon, teflon, FEP, LDPE, HDPE, PPCO (polupropylene copolymer) are completely DMSO-compatible whereas those of polystyrene, ECTFE/ETFE are moderately DMSO compatible. Polysulfone, PVC tubings and polycarbonate materials are incompatible with DMSO hence should not be brought in contact with DMSO.
For filter sterilization of DMSO, teflon or nylon membrane filters are recommended. Cellulose acetate membranes should not be used."
I am working on a project involving cryopreserving cord tissue and currently both cord blood and cord tissue are exposed to DMSO for a maximum of 10 minutes before being frozen. We are now working to adjust the time for exposure to the cord tissue as we feel that 10 minutes is probably not long enough. Does anyone on here have data or experience of this?
My experience in my previous lab was that any drug dissolved in 100% DMSO is quite toxic to the mice when delivered over an extended period of time (such as once daily injections for several days). Even reducing the DMSO concentration to 50% is still pretty toxic, but DMSO concentrations of 10% or below seem to be okay. I would recommend testing the solubility of small amounts of your compound in other vehicles; a couple that have worked for us (and been less toxic to the mice) are listed below:
I am using DMSO soluble compound to treat HepG2 cells. The problem is when I dilute DMSO soluble compound in water to get the desired concentration for treating cells, I am getting precipitation of the compound. Does anybody have experience working with DMSO soluble compound in cell culture?
The best way to handle solubility problems, is to keep the DMSO concentration as high as possible in the final assay, and dilute directly from your DMSO stock into the cells with media. Also it is critical to make the serial dilutions in DMSO if you are doing a dose response. So let's say the cells can take 0. 1-0.2% DMSO as a final concentration. Then if you have 1 ml volume, you can add 1-2ul of your compound directly into the 1 ml.
I don't remember what the maximum concentration your cells can tolerate, but I would use that concentration and work backwards to determine how many ul you need to add. This is really the only way to deal with insoluble compounds.
So if your final concentration is 1 uM, then I would add 1ul from a stock of 500uM in DMSO only, to 500ul of media with cells. Or you can try to make a 10% solution of DMSO with a 50 uM stock, and add 5ul. If you make the 10% stock, it is risky, but you can see if it's precipitates. If your cells can tolerate more DMSO than 0.1-0.2%, then you can add more ul of a more dilute stock solution.
Does anyone have experience of a reaction of NHS-activated carboxylic acid with an NH2 bearing molecule in DMSO?
Nov 18, 2013
I have poor water soluble peptides and I am trying to exploit the NHS/NH2 chemistry in DMSO. I tried in a mixture DMSO/aqueous buffer, but I have solubility issues.
I never had anything so insoluble that wouldn't go in DMF. I'd try to stay away from DMSO, it's very hard to get rid of it afterwards, and it will mess your HPLC retention times (probably will drag most of your peptide through the column). I'd try to play with the pH, DMF, acetonitrile/MeOH,..
Have you considered making the modification in the solid phase before cleavage?
Because DMSO act as vehicle therefore didn't effect the the aortic relaxation and contraction depending on the concentration that you used for the preparation of the extract, it is important that the concentration of DMSO not larger than 5% of solution inside tissue bath
I need to do anticancer activity and some biochemical parameters of plant extract in mice, my extracts dissolves well in pure DMSO. Is it safe to use pure DMSO as a vehicle if not, what percentage of dilution is normally recommended?
I have a problem in preparing some ruthenium complexes. I tried to prepare some Ru complexes and I failed many times. Elemental analysis does not match. I don't know what is the problem. I succeed to prepare Ru(DMSO)4Cl2 as a precursor complexes and then try to replace (DMSO) with other ligands after reflux for certain times by addition of some reagents. I could precipitate a complexes, but when I do elemental analysis for it I found that there are no match between calculated and found data. I don't know what is the reason for that. I tried many times but I failed in all of them. Can anyone help me please ?