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Does anyone have any experience using the membrane-permeable acetoxymethyl
ester derivative of NITR-5?  I've been loading it into HeLa cells and,
using flash photolysis, trying to generate calcium transients equivalent
to those stimulated by a PLC-activating agonist (e.g. histamine).
Unfortunately the peak calcium release I've been getting has been somewhat
weak - less than a fifth of that evoked by 100 uM histamine addition.

Are there any tricks to using this compound?  Are there any alternatives?

I've been loading the NITR-5/AM at 20 uM for 35 min at room temp in a
HEPES buffer (pH 7.4) containing 1 mM Ca2+, then leaving to de-esterify
for 35 min before imaging.  Cells are simultaneously loaded with Fluo4/AM
at 1 uM.  I'm using a MicroPoint flash photolysis system on a Zeiss 510
META, and am confident that the flash settings are releasing most of the
loaded cage as a subsequent flash has little effect on [Ca2+]i.

Any advice would be greatfully received.
Simon