From: Alice L. Givan (Alice.L.Givan@Dartmouth.EDU)
Date: Mon Sep 27 1993 - 09:18:58 EST
The best way that I know around the problem is to make a standard curve with a range of beads that have been calibrated in terms of equivalents in soluble fluorescein (ESF). Run the beads at the voltage and gain settings that you will be using in the experiment. Plot a curve of median fluorescence intensity on one axis against ESF on the other axis for these beads or calculate the regression line equation. Then you have to run the cells through the cytometer to get the median fluorescence intensity of both cell lines unstained and both cell lines stained. By using the standard curve or the equation, you can convert the fluorescence intensity of all four conditions of cells into equivalent soluble fluorescein molecules. And then you can subtract the control values from the stained values for each cell line and get an expression for each cell line for the relative number of specific antigen molecules per cell ( expressed in terms of equivalents of soluble fluorescein). If you need to use different voltage or amp gain settings for the two cell lines to get them on scale, you can run the beads at the two different settings, plot two different standard curves, and calculate the ESF values for the two cell lines according to the relevant curve for each. Any more straight-forward methods?? Alice Givan Alice.L.Givan@dartmouth.edu NCCC Flow Cytometry Laboratory/Department of Physiology Dartmouth Medical School Lebanon, New Hampshire 03756-0001 USA voice 603-650-7907 fax 603-650-6130
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