From: Andrew D. Wells, Ph. D. (adwells@mail.MED.UPENN.EDU)
Date: Thu Oct 29 1998 - 17:49:34 EST
>We are also currently trying to stain mouse splenocytes with >CFSE dye (cells are stained with 2.5-5 uM final conc. dissolved >in DMSO final <0.1% in PBS for 8-10 min @ room temp. and >washed 3-5 times with 10% serum medium ) to analyse cell divisions >of CD4 cells and intracellular cytokine production, but with less success. >We have a number of problems in the assay. The main problem was >that splenocytes stained with CFSE did not respond to stimulation with >either ConA or PMA/Ionomycin or in some instances tend to die in >culture. Did anyone else have this problem in CFSE staining assays? >We would appreciate if anyone can give any suggestions to get around >this problem. We also had the problem of compensating FL2-FL1 >to distinguish different cell generations (peaks). Does anyone have >any suggestions for compensation of FL2-FL1 or do we have to try other >flourochromes such as Red670 and TriColor (FL3 on FACScan) for >other staining. > >Thanks in advance. > >Sathi >UMass-Amherst Sathi, Have you experienced probems in priming and/or detecting intracellur cytokines in unlabeled T cell populations, or do these problems occur only when attempting to use CFSE-labeled cells? If you have worked out the proper control stimulatory conditions, then in my experience, an inability to detect normal lymphocyte function (or to obtain defined CFSE peaks) reflects a problem during the initial CFSE labeling procedure - I'm a little confused with your labeling procedure described above. We use a procedure modified from the original Lyons & Parish protocol, starting with 5 millimolar (mM) CFSE (Molecular Probes #C-1157) in DMSO. We've been diluting this 2000- to 4000-fold to a final concentration of 1.25-2.5 micromolar (µM) in PBS. The cell density should be less than 5 million per mL and you should limit the duration of labeling to 3 minutes to avoid excess clumping. To quench the labeling reaction immediately, we add serum to 20% final volume and wash twice in PBS. We usually lose approx. 30% during this labeling procedure - the remaining cells are viable and as responsive to various T cell stimuli as unlabeled splenocytes. We have also observed no greater propensity for CFSE-labeled cells to dye during stimulation compared to unlabeled cells, as judged by vital dye exclusion. 24 hours after labeling as described above, you should be able to compensate the CFSE signal out of FL2 with no problems. We have been able to obtain very good IL-2, IFN-g and IL-4 intracellular staining by restimulating 2- to 5-day primed, CFSE-labeled cells with either PMA and Ionomycin, immobilized CD3/CD28 Abs, or peptide-pulsed spleen cells (when using TCR-tg T cells) for 4-6 hours in the presence of 2 µM monensin. A similar example of the detection of cytokines in CFSE-labeled T cells by intracellular staining can be found in a paper by Bird et al., Immunity 9:229, 1998. If you optimize your labeling procedure and keep close tabs on how many cell you're losing and how many you're plating, I think you'll get it to work. Best wishes, Andrew Andrew D. Wells, Ph.D. University of Pennsylvania Department of Medicine 904 Stellar-Chance Laboratories 422 Curie Boulevard Philadelphia, PA 19104 (215) 898-1951 (215) 573-2880 (FAX) adwells@mail.med.upenn.edu
This archive was generated by hypermail 2.1.6 : Thu Jan 01 2004 - 17:35:26 EST
