Fellow Flowers: Dagna Sheerer reminded me of a recent query by her along these lines (Jan,2000). (I KNEW I had seen it- but some how my search of archives came up empty). I especially laughed when I found MY response in there! Typically, I freeze in 90%NCS/10%DMSO. With these conditions, I've had nary a problem. I thaw (fast),spin, wash2x in regular RPMI/10%NCS or PBS/2%NCS and count w/Trypan. No problems. But these samples were frozen under questionable circumstances. So it was not clear to me if the thawed sample conditions were related to the originalfreezing conditions, thaw technique or both. Well, now a new wrench- several people have pointed out that using Trypan on DMSO cells are the problem! That the membrane is "leaky"; so in fact they may really have a much higher viability. Of course this makes perfect sense, although in 90% serum I use trypan and have good dye exclusion. Go figure! So, for my samples that I'm only doing FACS on- I'll just use PI. It's not as convenient,though, as a quick check on a scope. [Our FACS is cross-campus, so I can only do PI at time of analysis]. We like to have a rough idea when we set up staining (it helps me decide if to try any CD14 markers or not, in case TFTC). Please excuse the heresy, but: Can anyone point me towards a NON-FACS PI-type viability stains that one can use on a simple (non-fluor)scope? [This is also helpful when we set up cultures for EliSpot and do not have time to incorporate a FACS analysis prior to culture prep.] Thanks for all the emails-I really appreciate it. Since the advice I rec'd really mirrors Dagna's post from Jan, I won't repost a summary. But if anyone would still like the emails forwarded-please ask & I'll be glad to share. 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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