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>We are currently analysing rat peritoneal macrophages for surface=20
>membrane receptors after staining with fluorescein-conjugated ligands.
>"Resident" macrophages are often larger than 60 =E6, thus considerably larg=
>er=20
>than the 20=E6x64=E6 laser beam at excitation focus of our FACScan & FACSor=
>t=20
>flowcytometers. The fluorescence intensity from the cells suggestedly=20
>mirrors the receptor density at the exposed part of the cell membrane,=20
>but hardly reflects the total receptor pool in these cells, since=20
>variation in surface area might contribute. Comparison with binding of=20
>radio-iodinated ligands does not exhibit convincing correlations.
Are you looking at FL1-Area or FL1-Height measurements? I would think that
with cells larger than the laser beam width, the Area signal would more
accurately measure the total fluorescence for each cell, where as the
Height signal would only measure the maximum fluorescence of a "cross
section" for each cell (not even the max. fluorescence for the entire cell).
(I think this is what you stated). FL1-Area should be available from the
doublet discrimination module (DDM).
>It might be common knowledge, but can someone give me a clue to=20
>correction for surface area by arithmetic on FSC, or would it be far=20
>reached to introduce a "topical intensity times FSC" parameter in huge cell=
>s?
Your FSC signal might not be giving you an accurate measure of "cell size".
Since you are approaching a slit-scan system, perhaps a Width signal would
better measure "cell size"? You could use the FL1-Width signal from the DDM
in the manner described by David Galbraith (see reference below).
There is a technique for measuring surface receptor density (see references
below). It is computed using a ratio. The FACScan cannot compute ratios.
The only program I know of that will compute ratios (and other things) is
(Bob Murphy's?) CALC4 program, which came as part of BDIS' Consort VAX
(a.k.a. Consort 40). Maybe someone else knows of another program?
Galbraith DW: Isolation and flow cytometric characterization of plant
protoplasts. In: Methods in Cell Biology, Volume 33: Flow Cytometry,
Darzynkiewicz Z, Crissman H (eds.), Academic Press, Inc., San Diego,
1990, pp. 549-562.
Shapiro HM: Practical Flow Cytometry, Second Edition, Alan R. Liss, Inc.,
New York, 1988.
Note: a newer 3rd Ed. was published in 1994. See a "Short Summary" of this
and other texts on the Purdue web server:
http://www.cyto.purdue.edu/flowcyt/refgen.html
Shapiro sites the following references for receptor density:
Matsui Y, Staunton DE, Shapiro HM, Yunis EJ: Comparison of MHC antigen
expression on PHA- and MLC-induced T cell lines with that on T and B
lymphoblastoid cell lines by cell cycle dependency. Human Immunol 15:285
(1986).
Matsui Y, Shapiro HM, Sheehy MJ, Christenson L, Staunton DE, Eynom EE,
Yunis EJ: Differential expression of T cell differentiation antigens and
major histocompatibility antigens on activated T cells during the cell
cycle. Eur J Immunol 16:248, (1986).
/\/\/\_ Eric Van Buren, vanburen%flovax.dnet@rocdec.roc.wayne.edu
\ \ \ Immunology & Microbiology
\_^_/ Wayne State University, Detroit, Michigan, USA
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