Volker, I'd just like to add that my experience with PBMC agrees with Randy's. However, you can have the odd problem with mouse IgG1 and human PBMC donors who are homozygous for the R131 polymorphism of Fc gamma RII (so called high-responder). mIgG1 binding can occur on these cells and result in an unexpected elevated background. Statistically this should be about 1 in 4 but I rarely saw this many significantly high backgrounds, which suggest that perhaps receptor density also was an influence. Craig Turner Senior Scientist CDL NBS ---------- From: fischer1@mail.nih.gov To: cyto-inbox Subject: RE: Fc-Blocking Date: Friday, April 12, 2002 9:04PM Volker, The answers to your questions are not as simple as you might like. 1. Blocking: Depends. We have found no significant difference using mouse IgG1 antibodies to stain human PBMC with or without blocking. When we use mouse IgG2a/b, we must block with rat serum or we get higher backgrounds. And when we do staining that requires secondary antibodies that are goat or rabbit (yes, some of our antibodies still require secondaries), we find it necessary to block with those sera as well. 2. How fast/how long: Again, I am sure my opinion is one of many different ones you will encounter. But, we stain 100,000 cells and I routinely recover at least 80% after multiple washes and fix/perm procedures. Our speed/force is 450xg for 10 minutes if using 15-50mL tubes. For the 12x75mm tubes we spin at 450xg for 5-6 minutes. 3. Buffers: Well, here is that black box of cell biology. We all know from years of experience/dogma that cells require protein to keep them "happy". However, I have used straight DPBS without Ca/Mg for staining human PBMC and the cells did fine, but it was a quickly done test that required minimal time. So, we routinely use the DPBS with 1% BSA to provide a little extra buffering to keep the cells happy in case we have to leave them a little longer than expected, or if it is one of our multiple step staining procedures. Good luck, Randy T. Fischer NIH/NIAMS Building 10, Room 6D57 9000 Rockville Pike Bethesda, MD 20892 (301) 594-3537 fischer1@mail.nih.gov > ---------- > From: Eckstein, Volker > Sent: Thursday, April 11, 2002 7:28 AM > To: Cytometry Mailing List > Subject: Fc-Blocking > > > Hi flow-experts, > > Fc-blocking (or not) on human PBMC prior direct or indirect staining is a > never ending discussion or story in our lab. > And the literature is confusing. > Some claim to use an IgG solution of the same origin as the used cells > are. > Others an IgG solution of the same origin as the monAb (direct coupled > with > fluorochromes) or the secondary polyclonal Ab are. What is the flow > community thinking about that issue and is there really a need? > > Some more questions: > What is the best wash and staining buffer for human PBMC staining > protocols? > Some people add BSA or HSA and some FCS in different concentrations (0.1 - > 5%) to a PBS-buffer. > And what is the cell biological need to add serum components? I suppose > not > blocking Fc-receptors. The explanation I've red that cells feel at home > and > comfortable is unsufficient (for me). > > And finally the question of speed in centrifugation of the cells for > washing > after staining. > Mostly published protocols differ extremely. The range in speed starts > with > 200g up to 1800g or more for 1 to 10 minutes. It depends on the source of > cells that's clear but even for human PBMC there are such differences > outlined. PBMC's are hard taking cells but where is the balance between > cell > loss by damage (high g) or cells loss by aspirating due to less pelleting > of > the cells (low g)? > > All responses are highly welcome. > > Best regards > > VolkerVolker Eckstein PhD > Dept. of Internal Medicine V > Medical School of the University > University of Heidelberg > GERMANY > volker_eckstein@med.uni-heidelberg.de > > > > > >
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