>Dear Flowers, > > I have come across a term "mean fluorescence index" in several >recent papers. This term is used to describe the total fluorescent >capacity of a population of cells and is derived from the percentage of >cells staining positive for a marker multiplied by the fluorescent >intensity of the population expressing the marker. Does anybody have an >idea about the acceptiblity of this calculation to express flow data ? > >-- >Sincerely, > >Harry D. Dawson, Ph.D. >Laboratory of Immunology >National Institute on Aging, NIH >5600 Nathan Shock Dr. >Baltimore, MD. 21224-6825 > >Phone: (410) 558-8300 ext. 7660 >Fax: (410) 558-8284 >E-mail: dawsonh@grc.nia.nih.gov I have seen this type of calculation suggested in applications that were functional in nature: calcium flux and oxidative burst. In calcium flux assays, the indo-1 fluorescence ratio is used as a measure of the concentration of intracellular calcium. In oxidative burst assays, the DCF fluorescence intensity is used as a measure of the concentration of intracellular hydrogen peroxide. In both assays you can calculate the "percentage of cells staining positive for a marker" (or "percent responding cells") and the fluorescent intensity (or ratio) of just the responding cells. By multiplying these two numbers together, you should get a kind of average fluorescent intensity (or ratio) of the entire sample (both responders and non-responders), which in turn would correlate to an average concentration of intracellular analyte, or perhaps a total amount of analyte per sample. I suppose you could call this the "total fluorescent capacity". This "mean fluorescence index" should be comparable to similar measurements made by spectrofluorometry, where the fluorescent intensity (and thus the concentration or amount of analyte) of the entire sample (not each individual cell) is measured. Using a "mean fluorescence index" calculates one number from two measurements. In doing so, however, some information is lost. If sample A has 1% responding cells with a mean intensity of 1000 units (index = 0.01 * 1000 = 10), and sample B has 100% responding cells with a mean intensity of 10 units (index = 1 * 10 = 10), such that both have an index of 10, are both samples the same? Both samples may have the same total amount of intracellular Ca2+ or H2O2 for the entire sample, but individual cells within each sample function differently. Although I guess it would be "acceptable" to report "mean fluorescence index" (especially if it were validated by comparison to a parallel method, like spectrofluorometry) for these assays, I would prefer the additional information present in reporting the percentage and mean (unless the index was shown to be more biologically relevant). After all, that is the true power of flow cytometry, the simultaneous measurement of several parameters on individual cells. Eric /\/\/\_ Eric Van Buren, aa9080@wayne.edu \ \ \ Karmanos Cancer Institute and Immunology & Microbiology \_^_/ Wayne State University, Detroit, Michigan, USA
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