Intra Cellular signaling by flow smonard at
Tue Dec 22 10:30:37 EST 1998

Hi Phil

We recently published a paper on this sort of thing in J.Exp.Med, Either
this month or last month. The first author name is Zhiwei Chen (sorry too
lazy to dig out the actual reference). I went for the Fluo3 Fura red
combination, its far better than fluo3 alone. We did it on a benchtop
machine (Calibur), On the Calibur I would use FL3 to collect to fura-red
signal and FL1 for the fluo3, if I had a sorter with a UV laser I would
certainly have used indo-1. The details are in the paper.

I have found the flux from different cells in response to chemokines is
quite variable. You get a good response with ionomycin so your cells are
loaded OK, probably the lack of decent sized flux is due to either your
cells being in bad shape, or your cells have responded to something in the
media or rough handling and are unresponsive (cells all fluxed out) or your
surface levels of chemokine receptor are low or your agonist is too weak.
Sorry a lot of "or"s.

If your cells are fluxed out a good rest for half an hour should help or
maybe change the buffer they are suspended in, we use HBSS without the red
stuff. I assume of course that you have calcium in the medium!  We would
get variable results with different cell types. Sometimes the mean ratio
would increase as little as 1.5 times sometimes up to 4 times.

Simon Monard

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