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
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