Flowers- I am in dire need of suggestions re: PBMC thawing protocols. I have approx. 100 PBMC samples that were frozen several years ago. I am attempting to thaw & stain for various cell surface markers. My sample recovery has been dismal, to say the least. The samples were prepared by personnel that have since left; and very little information has been provided to me as far as WHAT is actually in each vial (number of cells & such). So based on what we predicted would have been in each vial, I'm only recovering 10-20% of the cells. Even more depressing is that the viability of the cells recovered is usually 10-20%. I've researched freezing/thawing procedures until my head is spinning. I typically freeze samples in 90% FCS/10%DMSO, and have 70-80% cell recovery, 80%+ viability. It appears these archived samples were frozen in RPMI/20%FCS/10%DMSO. [This has been a popular method in the literature, nothing unusual]. I thaw by swirling in 37 water until partial thaw, add 1ml cold RPMI/10% to vial. Transfer contents to 15ml tube containing 1ml FCS. Rinse vial with another ml RPMI/10% FCS. Then QS to 5ml with RPMI/10%FCS. Spin & wash again in same. Then wash in PBS/2%FCS, and stain for FACS. [All steps done on ice] I check viability after the first wash-and already >80-90% are trypan blue+. Am I missing something? Can anyone point out a glaring mistake here? Another observation: I occasionally get samples where under the microscope the PBMC's look shrunken. No debris, no clumps, just really small. And they don't really look trypan+ (hard call though). When I run FACS- they stain beautifully (meaning good CD3/4 or 3/8 stain profiles- no "smearing" or nonspecific), but the FSC is shifted waaayyyyy down left-for ALL (lymphs/monos). Most of the notations I found in various archives discuss freezing methods. Since I can't fix what's already done, can anyone point me towards some crucial steps in thawing? 1) What is the recommended temp? Since I'm not culturing, should I do all on ice? (I've seen ref's where all steps are 37) 2) Do I have enough protein in my mix? 3) Is the change from 20%FCS to 50%FCS and back to 20% a concern? 4) Can viable cells be trypan-leaky? [Sometimes I see blue cells, but they are not the typical swollen/blue; and the FACS staining will look perfect]. 5) How much exposure to DMSO is too much during thawing? I try to thaw 2-3 vials at once. Bad idea? Please email me any hints/tips- I will post a summary of the advice for interested parties. Thanks! Bunny Bunny ******************************************************* Bunny Cotleur +*+ Bunny Cotleur Cleveland Clinic Foundation *+* 2001 Lester RD Neurosciences NC30 +*+ Valley City, OH 44280 9500 Euclid Avenue *+* 330-483-4800 Cleveland, OH 44195 +*+ bunny@cotleur.com 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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