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>Can anyone direct me to publications in which the time kinetics of
>binding of directly fluorochrome-conjugated antibody to living cells
>were followed by flow cytometry? The idea is to add the antibody to
>the cells and then immediately begin acquisition, without rinsing.
>Ideally, a single large file can be acquired over several minutes
>while binding proceeds; then medians can be taken on sequential
>"slices" of the data in the file. Thus, the entire kinetic curve is
>generated from a single sample.
>I'm particularly interested in the unrinsed mode described above, but
>I'd also be interested in any publications using a more conventional
>approach in which binding is stopped by rinsing at various times in
>different samples, and then each sample is analyzed as a single time
>point.
>Finally, I'd like to know what software is available (commercial or
>otherwise) to slice up the events in a single list-mode file. (I have
>some home-grown software under development if anyone is interested in
>using it.) Do any of the commercial packages (e.g. from Phoenix, Verity,
>B-D, or Coulter) do time-slicing?
The references below review the types of measurements Eric described,
give references to original work by Bohn & Mmanske, Crowell & Salzman,
Finney & Sklar and others, and give protocols for both equilibrium
methods and dilution methods. The KINPRO program in the Consort/VAX
package calculates gated means and standard deviations for time-slices
of FCS files and creates a file for plotting of these means versus time.
If CHRONOS is still supported for the HP, it does similar things.
R. F. Murphy (1990). Ligand Binding, Endocytosis, and Processing. In:
Flow Cytometry and Sorting, Second Edition, M. R. Melamed, T. Lindmo, M.
L. Mendelsohn (eds.), Wiley-Liss, Inc., New York, pp. 355-366.
R. F. Murphy (1992) Scatchard analysis by flow cytometry. In: Flow
Cytometry and Cell Sorting, A. Radbruch (ed.), Springer Verlag, Berlin,
pp. 59-62.
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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
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