Michelle Fiordalisi wrote "I calculate % positivity using BD's formula: (Activ. sample - activ. isotype control) - ( Unstim. sample - Unstim. isotype control)." I would be interested in other intracellular flow-ers opinion of this formula: do you use it widely? I have a problem with subtracting the unstimulated sample values. I presume tnat this is intended to allow for non-specific intracellular staining that can occur after permeabilisation. However, in some ex vivo situations where effector lymphocytes are already activated a small percentage of even unstimulated cells may be clearly positive relative to their isotype control. I doubt that it would occur in peripheral blood lymphocytes but I have certainly seen it for IFN-g in activated BAL cells in pulmonary disease. It occurred in situations where the clinical disease was highly active. By subtracting this percentage of cells from that in the stimulated sample one might be underestimating the total % of cytokine-expressing cells. What do the erudite people think? Dearbhaile O' Donnell, University College Dublin
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