Re: A FACS related problem
Alice L. Givan (Alice.L.Givan@Dartmouth.EDU)
27 Sep 93 10:18:58 EDT
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