I have a question regarding an experiment which our laboratory performed to detect CD40 expression levels on microglial cells before and after 24 challenge with CD40 ligand. This is the protocol that we used: Flow cytometric analysis. CD40 expression was assessed by FACS analysis. Primary cultured microglial cells were plated in 6-well tissue-culture plates (Nunclon, Nalge Nunc International, Denmark) at 2x105 cells/well and incubated with CD40L protein in the presence or absence of anti-CD45 mAb. Twenty-four hours after incubation, microglial cells (approximately 1x106 cells) were re-suspended in 250 microL of ice-cold PBS for FACS analysis, according to methods described previously (5). A minimum of 10,000 cells were accepted for FACS analysis. Cells were gated based on morphological characteristics such that apoptotic and necrotic cells were not accepted for FACS analysis. Percentages of positive cells (CD40 expressing) were calculated as follows: for each treatment the mean fluorescence value for the isotype-matched control antibody was subtracted from the mean fluorescence value for the CD40-specific antibody. We found that naive (untreated) microglia expressed ~4.5% of CD40 on their surface using this protocol, and following 24 h treatment with CD40L, this expression level rose to ~14.5%. The specific question is, do these percentages REALLY mean that out of 100 cells, x percentage express CD40? I tend to think that many more of those cells actually express CD40 but that such low expression levels of CD40 are undetectable by FACS. If this is the case, then what do these percentages really mean, and is there a reference(s) for this? Best wishes, Terrence Town Roskamp Institute University of South Florida, Tampa, FL 33613 Tel: (813) 974-3722 Fax: (813) 974-3915 email: ttown@hsc.usf.edu
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