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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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