We're conducting flow cytometry on human blood that has been separated into two fractions using FiColl -- a buffy coat layer containing mostly PBMCs and a red layer enriched in granulocytes. While the non-specific binding of our isotype controls in the buffy coat fraction looks okay, the non-specific binding in our granulocyte enriched fraction (which undergoes erythrocyte lysis) is much higher to the point where it's hard to delineate populations. We're currently using a human Fc receptor block, but I've been told that monocytes and some granulocytes have very high non-specific binding that cannot really be affected by Fc block. Are there alternatives, like normal mouse IgG or mouse serum (all antibodies are mouse anti-human)? Our flow cytometry wash buffer is PBS with 0.5% BSA and .03% sodium azide.
I tend to block with 20% human AB sera (15 minutes at room temperature); this also permits you to conduct staining for CD16 and CD32. You could also try staining in a higher percentage solution of BSA or FCS (5% opposed to 0.5%).
Thanks Sarah! Do you buy yours commercially or use heat inactivated from a health donor?
I don't heat inactivate the AB serum. I buy it commerically, make 5ml aliquots and freeze these until required.
We switched to 10% human serum and increase the BSA in the washing buffer to 2% and our background noise decreased significantly. Thanks for the advice!
I take advantage to ask: are we obliged to heat inactivate the serum (for exp. normal goat serum) to use it in flow cytometry staining protocol ?
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