We routinely perform 3-color flow analysis (FITC, PE and PE:Cy5) on our FACSCan. We have recently noted that while some commercial PE:Cy5 conjugates bind 'non-specifically' to monocytes and to a lesser extent granulocytes, and (presumably) B lymphocytes, others do not. It is well known that Cy5-conjugates (and thus PE:Cy5 conjugates also) bind to Fc gamma receptors (CD64) on such cell types and this probably explains thes observations. It has been suggested that one method of blocking this CD64-related problem is to add a Cy5-conjugate of some irrelevant protein, to the PE:Cy5-antibody conjugate. Since Cy5-conjugates are not excited by argon lasers, the blocking conjugate will not pose a problem. However, we also run samples through a FACSVantage sorter equipped with Enterprise and HeNe lasers. Thus, I assume that the use of such blocking reagents would not only be of no benefit in this setting, but also could cause additional problems. Does anyone have any better ideas regarding this issue. Rob Sutherland, Oncology Research, The Toronto Hospital
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