I would like to add a few comments to the recent discussion on CSFE labeling lymphocytes for proliferation analysis. 1. We adopted the protocol for bovine PBMC and have done quite an extensive analysis of differential responses of lymphocyte subpopulations in response to stimulation by a variety of mitogens ( ConA, PMA/Ionomycin, Anti-CD3 antibody, Anti-delta antibody and r-IL 2). The CSFE labeling system worked very well for our analysis and gave us valuable information about which sub-population are responding primarily, which ones respond as by-standers, what proportion (%) of each sub-population is dividing (at each cell division level) and also allowed us a time course analysis of response. All these differences were not detectable by the conventional 3H tritiated thymidine incoporation assay. I have attached a MS powerpoint file of a portion of one such experiment for a preview of those who are interested. 2. We prepared CSFE as a 20mM solution in DMSO and froze ~10-20 ul aliquots at -20*C. We were using it for more than an year now with out any detectable change in labeling activity. We use a dye dispersing agent called "Pluronic F127" (Molecular probes) supplied as 20% solution in DMSO, to help loading of CFSE very efficiently. We thaw out a 20 mM CSFE vial and mix with Pluronic F127 at 1:1 ratio, and then dilute the mixture in Hanks Balanced Salt Solution to 1.5-3 micro molar (uM) concentration and filter sterilize it before labeling. Use of Pluronic F127 allowed us to use the CSFE at very low concentrations ( bovine PBMC were very sensitive to CSFE and started dying at or above 5 uM concentrations) and gave an excellent loading of the CFSE in to the cytoplasm. 3. We noticed that for first 12 hours in culture, the cells were very bright for flowcytometry analysis. After initial quenching of some dye out from cells, after 12 hours onwards we were able to compensate the green(FL1) and red(FL2) perfectly for our two color analysis. We use to set FL2-FL1 compensations between 90 and 100% in order to set the compensation properly. We also had to individually titrate all our FL2 (PE) antibodies initially for surface staining for phenotype analysis to have them in a viewable level. Hope this information will be useful for those who are using CSFE analysis. Good luck Sathi --------------------------------------------------------------------------- T. Sathiyaseelan Veterinary & Animal Science Dept University of Massachusetts- Amherst. Ph: 413-545 5543 sathy@vasci.umass.edu
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