Indo-1 loading
MK (LCMK@WEIZMANN.weizmann.ac.il)
Thu, 21 Dec 95 11:01:43 +0200
Hi,
You haven't stated the nature of the problems you had with the probe but I
assume you can't see it in the cells.
You definitely should try the "Pluronic trick" (1 - 2.5 microM, final).
Also, keep your medium serum as low as you can afford to and make sure
normal levels of Ca++ ARE PRESENT in it.
I don't think detaching the cells from your culture flasks (or dishes)
has anything to do with problems in loading Indo-1. What you should look
into is to what extent your cells express esterases. Low levels of esterases
mean Indo1-AM is not cleaved into Indo1 and AM salt, so it can come out of
the cells and the very little probe that stays in them will not give you
the emissions you are expecting.
I hope this is of help.
Moshe Kushnir,
Dept. Immunology,
WIS, Rehovot,
Irael.
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