On Tue, 21 Sep 1999, Faith Kaplan wrote: > > Here's a general question that I had...and hoped that if I share this > problem with hands on users of flow cytometers that I may get real > answers! > I have to decide between a FITC or an R-PE tagged reagent. My staining > was for an internal > fixed/permeablized cell marker. I was using a FITC affinity purified > anti-rabbit IgG (H+L) or > an R- PE affininty purified F(ab')2 rabbit IgG (H+L). When staining on > the FACSCalibur using a control cell line, I got the same % of positive > cells using either secondary. However, when I switched to a primary cell > culture, using the same primary antibody (which was a rabbit polyclonal) > and the same 2 secondaries I now got 75% pos with the FITC tag and 35% > pos with the PE. What can be causing this? How would I decide which is > the correct color? > > I would really appreciate any information that anyone could give > me...answers, other people to contact, web sites, or review papers, etc. > > My work address is Ontogeny > 45 Moulton St > Cambridge, MA 02138 > phone 671-876-0086 x 298 > email fkaplan@ontogeny.com > > > I look forward to hearing any responses! > Thank you in advance for your help, > > Faith Kaplan > > Hi Faith, Since the FITC-conjugated secondary is whole molecule rather than F(ab')2, it has the Fc portion, which may be binding to Fc receptors on the cells of interest. Did staining with the secondary alone show increased flurescence as well? AR __________________________________ Arthur Roberts Dept. of Medicine Robert Wood Johnson Medical School email: robertar@umdnj.edu phone: 732-235-7790 Fax: 732-235-7792
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