Dagna Sheerar wrote: > > Hello Everyone, > > Occasionally, I have to thaw some PBMC's for Flow. They are frozen in DMSO > in 1 mL aliquots. The thawing procedure is as described on the instruction > sheet that comes with the DMSO. I am wondering what kind of yeilds people > get when thawing PBMC's. Is it normal to recover only 50% of the cells? > These are rhesus PBMC's and are infected with HIV/SIV/SHIV. > > I would appreciate any information anyone could share. > > Thank you very much > > Dagna Sheerar > Wisconsin Regional Primate Research Center > University of Wisconsin - Madison > 1220 Capitol Court > Building #2 Room #215 > Madison, WI 53715 > 608 265 3796 > dagnas@primate.wisc.edu Are you freezing in 100% DMSO? We use 80% newborn calf serum/20%DMSO. Also be aware that there are MANY grades of DMSO. We use Tissue Culture grade (endotoxin tested) from Sigma. In my lab we usually recover 80-90% of the cells. How are you thawing? I've seen methods that tell you to add media dropwise (I call the slow method) and some say thaw fast. We swirl the vial in a 37 C watear bath until about half thawed. Then add complete media (whatever type you will culture in). Transfer to 15ml conical that has 5ml media in it already. Ad 1 ml media to vial 2x more times to flush out rest of cells, combine with original. qs to 10ml, spin 1500rpm 6minutes, decant, repeat & count w/trypan (to also check viability). Works for us! Bunny ******************************************************* Bunny Cotleur Cleveland Clinic Foundation *+* Neurosciences NC30 +*+ 9500 Euclid Avenue *+* Cleveland, OH 44195 +*+ 216-444-1164 *+* cotleua@ccf,org +*+ ******************************************************* When you do something, you should burn yourself completely, like a good bonfire, leaving no trace of yourself. (Shunryu Suzuki)
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