Hello Kathy
I am guilty of originally responding directly to you. After reading the
latest round of "emails" i feel compelled to chime in on the list.
As i had indicated in my original response to you, the CD3+CD8- gating
strategy includes the activated CD4+ cells as well as CD3+ double negative
cells. alternatives to selectively gate on the CD4 positive T cells,
including mario's suggestions sound good but are a little tricky and will
have to be validated for each specific system. the CD3+CD8- gating
strategy will obviously not work in every possible situation (especially if
you are studying in vitro activation of CD4 T cells in ALPS patients), but
it certainly provides a very quick, easy and practical solution to capture
the recently positive CD4 T cells (and the CD3+4/8 double negatives). i
think everyone would agree that this gate does contain the CD4+ T cells.
at the very least the negative gating method provides a very easy beginning
to see if the substance you are looking for is in the cells in this gate,
which in most instances (other than those alluded to earlier) will be
predominantly CD4 T cells. if you get what you expect you will have to
prove that the result is not do to contaminating 4/8 double negative T
cells. if you don't get anything you have not wasted any time developing
alternative CD4 gating strategies.
i would be curious to know if CD4 binding potentiates/inhibits signalling,
has this been investigated? or is it assumed that prelabelling with CD4
prior to stimulation has no effect on signalling?
alternatively, if you choose cytoplasmic CD4 staining is the signal bright
enough to cleanly separate the CD4+ from the CD4- T cells? either one of
these methods may work, but i am not convinced that they have been
validated to the extent that would preclude at least trying what is a much
easier but admittedly not perfect solution. personally i would try the
easiest method first and would not discount any suggestions.
good luck
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Dr. Maurice (Mo) R.G. O'Gorman
Assist. Professor Pediatrics
Northwestern Univ. Med. School
Director Diagnostic Immunology and Flow Cytometry Laboratories
The Children's Memorial Hospital
2300 Children's Plaza
Chicago, IL 60614
Ph. 773 880 3070 office
773 880 4361 laboratory
Fax 773 880 3739
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