DNA and cell surface staining

From: Michele.Nadaf@mcmail.vanderbilt.edu
Date: Wed May 09 2001 - 13:24:58 EST


Dear Flow-ers.
I have been approached by someone wanting to analyze cell cycle (using PI)
and simultaneous surface stains using CD21-FITC and a biotinylated HSA on a
dual laser FACSCalibur.  (488 nm and 635 nm lasers)  I see no problem with
the FITC ab, however am seeking a recommendation for the unconjugated ab,
perhaps APC? Also would there be any major diffuculties with using a
biotinylated ab with this kind of stain, i.e. non specificity and what
could they do to avoid these problems?  I have done this before with FITC
conjugated surface Ab after several tries to optimize the fixation
procedure, (either loss of surface stain or insufficient PI stain) finally
getting it to look nice. However, have not tried it with a 2nd Ab that was
biotin.  Any suggestions on protocols that have worked best for you?  Any
help with the previous questions would be appreciated.  Thank you.
Michele



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