Dear all, dear Mario, May I bring a small correction regarding CD4 expression ? Mario Roederer recently wrote as an answer to Bill Hyun.: >CD4 T cells have 50,000 molecules of CD4 per cell (25,000 antibody binding sites).< I completely agree with the number of CD4 antibody molecules per CD4 T cell (50,000) that we also measured using our Quantitative ImmunoFluorescence Indirect assay (QIFI, see Poncelet et al., Res. Immunol., 142 :291-98 (1991) and Bikoue et al., Cytometry 26:137-147 (1996) ). However, I remember the number of binding sites per cell was measured at 100,000 antibody binding sites per cell using monovalent Fab fragments ( see Davis KA et al., Cytometry, 33:197-205 and Hultin et al., same issue p123-132 ). In this case where CD4 antibody binding has been demonstrated bivalent , the number of binding sites per cell is twice the number of CD4 antibody molecules per cell. I would recommend using the number 50,000 CD4 antibody molecules per CD4 T cell as a control (this makes only one single point) and use calibrated bead sets for calibration (they most often provide 4 or more points at various levels ). The number 200,000 CD45 antibody per lymphocyte is also a rather stable value according to our experience ( see Bikoue et al, upper) and may also provide an other (high level) control value. Hope that helps. Philippe Poncelet, PhD Director R&D BioCytex Marseilles France
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