The use of calcium ionophore, ionomycin, and calcium ATPase pump inhibitor thapsigargin on cells loaded with Indo-1 can be used to monitor the movement of internal calcium stores by the nature of their different mechanisms of action. Ionomycin will only release internal calcium stores within cells (when resuspended in calcium free medium); under such conditions, Indo-1 will detect all stores of calcium released into the cytoplasm (Fig. 1A).
Thapsigargin has differential effects on cellular calcium ATPase pumps, irreversibly inhibiting the ER calcium ATPase pump while not affecting those of the plasma membrane. Thus, any stores of ER calcium released are detected by Indo-1 in the cytoplasm on route to the mitochondria (Fig. 1B) (7). Indo-1 can thus be used to quantify internal calcium stores other than from the ER by subtraction of the Indo-1 calcium flux profiles generated by ionomycin and thapsigargin shown in an overlay plot (Fig. 2).
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