All we know, centrifuging right after thawing the cells is harmful. But in the other hand, remaining the DMSO in the medium can hit the cells, too. So shall I remove the DMSO or it's better to avoid the centrifuge?
Dear Aref, A stright answer to your question is 100% YES !
It is always recommended to centrifuge the cells after thawing to remove DMSO. But befor that, you can thaw your cells containg DMSO and add it to 4ml of culture media in a 15ml falcon and then you can centrifuge it. To avoid damage to cells you can go for low rpm or g values, say 2,000rpm or 800g for 2 minutes only. Then you can discard the supernatant and resuspend the cells in complete culture medium. Don't ever risk to keep the cells in DMSO for one day in culture media as that is more apoptotic cells and in case of centrifugation at the mentioned settings, you will hardly encounter any cell death.
You can keep the DMSO with the cells for a day or so, as long as the final DMSO concentration is below 1% (the exact concentrations depends on the cell type). Higher concentrations can be toxic or induce differentiation (eg in many leukaemia cell lines).
Dear Aref, A stright answer to your question is 100% YES !
It is always recommended to centrifuge the cells after thawing to remove DMSO. But befor that, you can thaw your cells containg DMSO and add it to 4ml of culture media in a 15ml falcon and then you can centrifuge it. To avoid damage to cells you can go for low rpm or g values, say 2,000rpm or 800g for 2 minutes only. Then you can discard the supernatant and resuspend the cells in complete culture medium. Don't ever risk to keep the cells in DMSO for one day in culture media as that is more apoptotic cells and in case of centrifugation at the mentioned settings, you will hardly encounter any cell death.
I also agree with Mr Kumar. You may proceed with that to remove the DMSO from the medium.
Best Regards,
yes, centrifugation after thawing is essential. it removes DMSO which is toxic for cells.
I agree with Afshin Samali. When dealing with adherent cells, at least with the commonly used ones (e.g. HeLa, U20S, 293), it is not necessary to remove DMSO by centrifugation (which, indeed, may harm the cells). We usually plate thawed cells in the culture medium and change the medium after the cells have adhered (~3 to 18 hours). I cannot, however, exclude the possibility that some cell lines may be very sensitive to DMSO and require its removal from the medium.
It would have been helpful if you specified the cell type. Epithelial cells normally do not need centrifugation as mentioned by Afshin Shamali and Vladimir Joukov. Just be sure that quickly thawed vial at 37oC is "quickly" added to the culture flask containing medium. Gently mix and leave overnight at 37oC incubator. You will find happy adherent cells the next day :) . At that point replace DMSO containing medium with the fresh one!
@Vladimir- Dear DMSO is toxic even at a 2-3ul volume/100ul for cancer cell lines and this is evident from MTT assay as I have done that lots of times( a little cell death is there in HepG2 cells) so you can guess that how detrimental can be a 100ul DMSO. and I am also dealing with primary culture of stem cells, so trust me, even stem cells dont encounter any cell death after the specified centrigugation
@Fatemah- Dear 2000rpm for 2 minutes is sufficient to get a good pellet and you need to minimise the centrifugation time so you can try for 2 minutes. We are working on nearly 20s of cell lines and primary cultures and it always yield a good pellet after centrifugation at recommended settings..Hope it helps.!
We recently purchased some new cell lines from ATCC (all adherent lines) where all but one cell line were recommended to be spun down at 125 x g for 5-7 minutes in 9ml of complete media to remove DMSO prior to seeding into the flask. The cell line that didn't require removal of DMSO said to change the media in the flask the following day after the cells had adhered to the flask surface. This line also said that if you really wanted to you could centrifuge to remove DMSO before seeding. I personally think it's best practice to remove DMSO before seeding.
Dear Romena Qazi . actually I work with different cell types, but at the time I work on AGS, MCF-7 and T-47D
all these are adherent cancerous cell lines
Dear Ajay Kumar
I usually centrifuge at 1000 rpm for 5 minutes but I believe it's harmful, isn't it?
Dear Afshin Samali
my cells are stored in 10% DMSO containing medium in liquid nitrogen tank for 6 months
Dear Aref
I am agree with the response of Ajay. I will add only that during the thawing step, perform it as quickly as possible (by placing the cell aliquot at 37 ° C) and next add the 4 ml complete culture medium (or 7ml - a larger volume of medium is better -) to reduce the stress caused on cells, that which will facilitate their recovery.
Regards
JP Gomez
Dear Aref, as Jean stated, quick recovery is imprtant here, so try to reduce centrifugation time to 2-3 minutes and u will not encounter any cell death due to thawing step. 2000 rpm for 2 minutes is by far the safest one which we have optimized for maximum cultures.
Dear students,
To thaw large cells of insulin-dependent breast cancer cultures like MCF-7 and T47D you should take a 50-100 ml glass with distilled water warmed to +37 degrees and put frozen ampoules there on the way to laminar. Then you should wipe ampoules with alcohol-wet cotton and transfer cells - gently! - into prepared in advance flasks with 2-3 times excess of warm complete growth medium, one ampoule content to one 25 mm flask, and put cells into CO2 incubator for at least several hours. DMSO content from 1 ml stock in 10-20 ml growth medium will never be more than 1 percent which theoretically could cause diffentiation after 2-3 passages. This growth medium is replaced for the new one normal volume the next day usually, it does not matter after 10 or 20 hours.
MCF-7 of normal growing passage do not survive 10 min. centrifugation more than 1200g, though single cells stay alive. So, if one uses typical cell centrifuge with rotor R about 20 cm or bigger I can imagine how much might be left from just thawed culture at 2000 rpm. Congratulations to ACTC, they have good business for their stocks sale this way.
I don't add more than 7-8 percent of DMSO for freezing T47D and MCF-7, but I freeze in pure serum in which you can leave them with DMSO for half an hour without any harm (FCS can be from some bottle with > expiration date, not to feel pity).
About "DMSO being very toxic" MTT-specialist should remember the concentration of this reagent which is added at the final stage for cell membranes lysis. You are right, it is 100 percent, and even better if growth media is removed before - now cells are destroyed, you can go to count with reader.
However marvelous thing is not DMSO, cells thawing or rpm. The miracle is what are current professors-supervisers of so many students in different universities doing for their salaries given by States regularily. Or it is only stupid me who was sitting with each new student in laminar and over microscope every day for 3 months, and there are real clever guys who are sitting in completely different places working days...
After that I can only hypothetize these professors would know everything better than two guys with only 3-5 hundred impact...
Dear Aref,
don't hope to see much as within last 10 years I hardly published articles. Everything new and working what is done goes into patents, and what is not new for laboratory anymore is not worth-interesting to spend time to put into article frame and to send out... My laboratory and I were paid for vaccines and kits development, so we did this, not publications.
Best wishes,
E.F.
I have work with fibroblast and Hella cell lines. washing them with 1X PBS solution with decreasing concentration of DMSO , PEG4000 and increasing concentration of media finally centrifuging it at 3000 RPM for 1-2 Min on 4 degree C. The process continue till 3-4 cylcle wih 2 cycle of added DMSO at very low concentration and 1-2 wash without DMSO and 1X PBS only. This will help your cell to protect from osmotic shock
its not compulsory that you centrifuge and remove DMSO while thawing. But make sure that you change the media next day after thawing.
Dear Aref, freezing cells in 10% DMSO in full media or serum is standard practice. The cells can be stored in liquid nitrogen for many years.
The breast cancer cells are adherent so when you thaw up the cells, transfer the contents of the ampule to fresh media (1 ml of cells to 12-15 ml of media) and transfer to a flask. Allow the cells adhere overnight and change the medium the following day. The cells will be absolutely fine. Good luck with your studies.
Dear Aref,
Two things you can do to remove DMSO from freeze cells.
1. Thaw cells as soon as possible at 37 degree water bath and add sufficient quantity of complete culture media in which it grows, centrifige it at 1000rpm for 1-2 min and seed them in fresh culture dish. after attachment in the culture dish you can change the media one more time.
2. Or if it is a fast attached cell line (As like Sf29) , then you can do one thing after fast thawing transfer the whole content to a culture dish containing sufficient quantity of fresh media, allow the cells to attach to the bottom (normally takes 30 mins). then discard the media containing DMSO and fill it with fresh media. Again after 30-45 mins you can change the media so as to further minimize the DMSO effect.
Think this will help you out.
Dear Aref,
I usually thaw frozen primary cells quickly in 37C waterbath, and invert the content of the vial into 10 ml of cell culture medium prepared in 15ml Falcon tube, ad spin the regular way (1200rpm). Counting cells before and after centrifugation showed similar number.
Best,
Oxana
The actual centrifugation speed and duration varies depending on the cell type.
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