Dear FLOW Scientists, I have two questions which have been bothering me for a while. 1. Does the auto-fluorescence affect on the Quantitation of FL? -Case 1: Are the two different cells with the different auto-florescence levels are comparable? Example:HLA-DR expressions on B cells and Monocytes The Geo mean (GM) for the B cell population (unstained/isotype) is 5. The GM for the Monocyte population (unstained/isotype)in the same sample is 50. There is the quantitative linear equation based on the same instrument setting measured by standard quantitative beads at the same time. Can we ignore the difference between 50-5 by the auto-flourescence? If the GMs of positive population for the B cells and Monocytes are same, for instance, 500, the calculated FL molecules/cell for both populations are same. Or 495 and 450 are the GMs to use? (perhaps it is not so simple.) -Case 2: To monitor up-regulation of an antigen in vitro, the auto-fluorescence increase day by day along with the increase of FL intensity for the antigen (it really depends on cells, media, growth factor, etc.). Is it truly showing the increase of the FL by the Ab, or the increase of the FL came from the increase of the auto-fluorescence of the cells? In this case also, the calculated FL molecules/cell based on the quantitative linear equation concludes up-regulation of the antigen on the cells. 2. Does an Annexin-V negative population indicate the amount of bindings to phosphatidylserine molecules (PS)? The negative population sometimes shows a great amount of FL index compared with cells only or isotype control. Also the negative peak shift by a sample to sample, even though they are same cells (might be due to the staining technique). Are the shift by a sample to sample, or non-Annexin-V Ab sample to Annexin negative population show the difference of the number of PS? How much do cells naturally express PS on the surface? I appreciate your help. Please let me sleep well. Mayumi Kataoka FACS Facility Temple University Philadelphia, PA 19140 __________________________________________________ Do you Yahoo!? Y! Web Hosting - Let the expert host your web site http://webhosting.yahoo.com/
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