A Practical Guide to the Preparation of Ca Buffers - Methods in Cell Biology - page 24

sold commercially for this purpose, but they may not mimic your preferred con-
ditions (and we have not used them). Once your electrode or fluorescent indicator
is calibrated, you can use it to measure [Ca 2 þ ] in more complex solutions, where
solution predictions are less reliable. These more complex solutions could be a
series of solutions of di erent [Ca 2 þ ] or [Mg-ATP], for example, to activate

skinned muscle fiber contraction, expose to permeabilized cells, dialyze into cells
via patch pipettes or use directly in biochemical assays in vitro. This is certainly a
rational and practical approach. One practical caveat is that the a

V

nity of most
fluorescent Ca 2 þ indicators changes (usually decreases two- to fourfold) in the
cellular environment versus in protein-free solutions ( Harkins et al. 1993; Hove-
Madsen and Bers, 1991; Konishi et al., 1988; Uto et al., 1991 ) and this seems to be
due to the interaction of the indicators with cellular proteins (which can be
mimicked in vitro). So precise control and measurement of [Ca 2 þ ] i in cells are
both very di

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cult to fully achieve. On the other hand, the importance of [Ca 2 þ ]
makes it important to measure and try to control [Ca 2 þ ] as best one can. Aware-
ness of the limitations may seem daunting, but should not dissuade one from these
valuable experiments. Even relative [Ca 2 þ ] changes and imperfect control or
measurement of [Ca 2 þ ] are of value in understanding these processes.

Y

Acknowledgements

This work was supported by a grant from the National Institutes of Health (HL30077).

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