Gib, I have had similar problems with non-specific staining with 8D4 in human cells and I have seen similar posts such as your own. In certain situations this seems to be more of a problem. For antigen specific responses, I find the background staining of 8D4 to be higher than acceptable; for mitogen activation, it is probably not a problem. I think there is a general ignorance on the part of casual users such that they figure if it is sold as an mAb to IL-4 that is all it recognizes. Many investigators use 8D4 and like the results. I think the epitope it recognizes is more resistant to aldehyde denaturation and so they get better staining with 8D4 than with the other generally available clone, 25D2. This may be a more of a problem with commercial fix/lyse reagents than with fresh 4% PFA. Although all of this sounds a bit obsessive, the choice of IL-4 mAb can make a big difference in the quality of one's results. We recently did epitope mapping of all of the clones that are commercially available as PE conjugates (clones 8D4, 25D2, 3007, 3010). The bottom line is that 25D2 and 3010 cross competed each other as did 8D4 and 3007. Hope that helps. Calman > ---------- > From: Otten, Gillis > Sent: Thursday, February 14, 2002 20:59 > To: Cytometry Mailing List > Subject: IL4 intracellular staining > > <<File: InterScan_Disclaimer.txt>> > We just tried using Pharmingen's PE-conjugated anti-human IL4 (clone > 8D4-8, > recommended for nonhuman primates in the Fall 2001 issue of "Hotlines") > with > freshly isolated rhesus peripheral blood mononuclear cells that were > cultured overnight in the presence of Brefeldin A and the in the presence > or > absence of Staph enterotoxin B (SEB). Using 0.2 ug of antibody > (Pharmingen > recommends < 0.25 ug per 1 million cells) we observed equivalent staining > of > subpopulations of unstimulated and stimulated T cells (intracellular > CD3+), > and also observed equivalent staining of a subpopulation of CD3-negative > lymphocytes (non T cells). In contrast, when we used PE-conjugated > anti-IFNgamma or anti-IL2 in the same experiment we observed staining only > of SEB-stimulated T cells, as expected. Does anyone have any comments on > why we are not seeing T cell-specific, stimulation dependent IL4 staining? > > Gib Otten > Chiron Corp. > >
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