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Typically 5 microlitres whole blood (preferably citrated or ACD) is diluted in
100 microlitres PBS 7.2 and incubated with the antibody for 30 minutes at RT.
1 ML of 3% Ammonium Oxalate (in aqua.dest) is added to the tube at RT and the
RBC's allowed to lyse.
This time can vary from patient to patient but 5 - 10 mins is usually more than
adequate.
A minimum dilution of 1 in 20 is required for adequate lysis.
The lysis is self limiting and does not appear to affect PLTS (or WBC's) at all,
so lysing for a longer time is no problem.
A normal control assayed for CD41 will usually give greater than 98 % positivity
in the Platelet gate.
Dropping the Amm.Ox to 1 or 2% will increase lysis speed but will also increase
the debris/rbc ghosts in the gate.
If this approach is suited to your work I would appreciate any feedback of it's
behaviour with other antigens.
I shall also copy this to the list for yet wider feedback.
Wal Sharp
SQU
Sultanate of Oman
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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;
Web