Hi Everybody in Flow Land, I have a sorting dilemma which I hope someone can help me with. The problem I'm having is that I don't see any cells (flourescence or FS/SS) when checking the purity! I double checked the delay and phase which were correct. I thought the cells might be sticking to the tube so I spun them down and did a cell count. It was right on with what the cytometer said I had. I tried running these in a small volume 200ul but still did not get anything. Initially I was trying to reanalyze small numbers (<20,000 sorted cells). When I sorted 50,000+ cells, I was able to see some (1-10%) cells upon reanalysis. I tried doing the same thing using Flow-check bead with the same results. I did not see this problem with other sorts I've done where I've sorted anywhere from ~50,000 to 1.2 x 10^6 human lymphocytes into collection tubes. Anybody have any idea why I can't see the cells which are clearly there? INFO: I am sorting mouse splenocytes which have been separated on a CD4 MACS column. The cells are FITC/PE stained. The sorter I have is a Beckman Coulter Elite (with the ESP module). cells are freshly isolated and kept in media with 10% FCS Sort collection tubes contain media with ~10-20%FCS 1 drop sort used; tried various combinations of Coincidence abort and PPU Any help would greatly be appreciated!! Thanks in advance! Andy (:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:)-(:) Andy Oberyszyn, M.S. The Ohio State University Analytical Cytometry Laboratory 416 Comprehensive Cancer Center 410 West 12th Avenue Columbus, Ohio 43210 Tel: 614/292-FLOW(3569) Fax: 614/292-7335 E-Mail: cytometry@osu.edu (-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-):(-) "What if the Hokey Pokey is really what it's all about?!?"
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