Hi List: Hope this intelligent group can help! I have been studying marker "T" on monocytes. Originally I have been collecting blood samples in EDTA, but found that untreated cells would have a fluoresence intensity 5-8 times higher than the isotype control (which did not make sense at all). The treated cells appropriately show a higher fluorescence than untreated cells (fluorecence of treated: untreated cells = 3:1). That is, for example, isotype control of treated and untreated cells has median fluorescence of 5, while untreated cells stained with antibodies to marker "T" has median flurescence of 40, and treated cells stained with antibodies to marker "T" has median fluorescence of 120. After trying different concentrations / amount of antibodies, the fluoresence intensity of untreated cells lowered a little bit but was still high (about 3-5 times higher than the isotype control.) Then I tried using Na citrate as the anticoagulant, and now my untreated cells has a flouresence intensity close to isotype control--however my treated cells' fluorescence intensity also lowered, although still higher than untreated cells (flurescence of treated : untreated cells = 2:1). In this case, the isotype flourescence is 5, the median flourescence of antibody "T" in untreated cells is 6, and the treated cells' median fluoresence is 12. Does anybody know why this happens? And which anticoagulant should be used? Thanks for your response! Annie
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