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I addressed a reply to your query at the two addresses shown on your email
to the List, and both of them bounced back to me. I am now posting this to
the List as well. You can then reply to me as you wish, and hopefully we
will have a contact address that works for me too.
Dear Rob,
You may start by testing the process to see what happens to the NK cells'
Fluo3 signal vs time. If it is initially invariant, you're probably home. I
have no idea, but I would guess that the time frame for effector-target
interaction and the subsequent mobilisation of the calcium in NKs is longer
than the transit time from the sample tube to the excitation point on some
machines. You can in fact vary that time by changing the length of your
sample line, sheath velocity and sample pressure differential (careful).
In any case, on first mixing there should be some small(?) fraction of the
NK cells which have not started the interaction with the targets, because
they haven't had time to make contact. You can weight this effect in favour
of either cell type as well by starting with different excess ratios of
either the target or the effector cells.
Another other way would be to spike the sample with some Fluo3-loaded NKs
(say 1% of your added cells) that were labelled by a "receptor"-blocking
non-fluorescent antibody, known to block the event you are trying to
follow. Make sure you wash them free of unbound antibody before you mix
them, otherwise there may be equilibration with the unblocked effectors,
and it may bias the result.
Does any of your team there remember Dr Max Joffe? An immunologist with a
very dry wit, working now in Melbourne. We are friends. Do tell.
The first address I tried is below, and includes ".smtpgw." . The other
address I tried was pyle@kfshrc.edu.sa
Best regards, Bob Ashcroft
-----Original Message-----
From: Robert Pyle [SMTP:pyle@smtpgw.kfshrc.edu.sa]
Sent: Tuesday, June 03, 1997 12:45 AM
To: Cytometry Mailing List
Subject: Calcium Measurements
I have a problem with an experimental design that I hope someone might help
me with.
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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