The question is whether closely bound reagents are absorbing light from their neighbors ( there is a term for this)or are the antibodies competing for sites. I have seen cases of CD8 antibodies blocking CD3 sites. To solve the problem of competition you have to stain one antibody at a time. You do not have to wash between staining. In a three color stain you will have to try the different combinations. On the kappa, lambda 19 stain. Try 19 in fitc since it is the common on both kappa and lambda. this will cut down on the PE/PE competition. Jim Houston -----Original Message----- From: Tom McHugh [mailto:mchugh@pangloss.ucsf.edu] Sent: Thursday, September 21, 2000 3:05 PM To: cyto-inbox Subject: steric hindrance? With 3-color we see significant reductions in staining intensity in the PE-Cy5 or PerCP-Cy5 signal (FL3) when there is also a strong PE signal. I assume this is steric hindrance. The antibody combinations of "kappa FITC, lambda PE and CD19 PE-Cy5 or PerCP-Cy5" and "CD10 FITC, CD34 PE and CD45 PE-Cy5 or PerCP-Cy5" show this reduction in the staining in FL3. We see this from different vendors and with a high % of samples which have the right antigen mix. Diluting the antibodies does not help eliminate this problem. Are others having this problem? Any practical suggestions as to how to avoid this? Thomas M. McHugh Technical Director, Clinical Laboratories UCSF Medical Center mchugh@pangloss.ucsf.edu
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