From: MK (LCMK@WEIZMANN.weizmann.ac.il)
Date: Thu Dec 21 1995 - 04:01:43 EST
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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