Andreas,
CA++ measurements by flow are fairly straightforward. Assuming your instrument setup is
correct (please be sure of this . . . UV laser, proper filters), the biggest problems
are with indo loading. If your detector voltages are relatively high (indo's quite
bright), you probably don't have good loading.
Viability is extemely important, of course. Dead cells don't flux . . .
Also, indo is a P-glycoprotein (MDR) substrate . . . if your cells are drug resistant,
you may not get good loading.
If these don't offer insight, an outline of your approach (inst. configuration,
loading procedure, buffers, whatever) may reveal the problem.
MAK.
>>> <andreas.simm@medizin.uni-halle.de> 01/25/02 16:47 PM >>>
Hi everybody,
we have a BD FACS-Vantage and have tried to measure
Ca-fluxes in GFP overexpressing cells. The cells used are
a low-adherent tumor cell line and were detached by
little mechanical force (tapping the flask). We used INDO
as the Ca-dependent fluorescent dye.
Unfortenately, we had bad luck. Even with ionomycin, we did
not get a Ca response.
Has somebody experience with this combination (GFP INDO)
and can help us?
A second question:
Who knows a flow cytometry company working about blood
homeostasis (platelet activation ...) in Europe?
Thanks in advance
Andreas
PD Dr. Andreas Simm
Universitaet Halle Wittenberg
Klinik fuer Herz- und Thoraxchirurgie
Ernst-Grube Str. 40
D-06120 Halle
Tel.: +49 (0) 345 557 2647
552 2878
FAX: +49 (0) 345 557 2782
552 2890
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