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Intracellular Calibration of the Calcium Indicator indo-1 in Isolated Fibers of Xenopus Muscle

Intracellular Calibration of the Calcium Indicator indo-1 in Isolated Fibers of Xenopus Muscle

H Westerblad, D G Allen

Biophys J. 1996 Aug;71(2):908-17.

PMID: 8842230

Abstract:

Estimates of the free myoplasmic [Ca2+] ([Ca2+]i) with fluorescent dyes are complicated by the fact that some properties of these dyes are altered in the intracellular environment. In the present study indo-1 was used to measure [Ca2+]i in isolated muscle fibers from Xenopus frogs. Fluorescent ratio signals obtained from indo-1 were converted into [Ca2+]i by means of an intracellular calibration method, which involved microinjection of 0.5 M EGTA and 1 M CaCl2 to get the ratio at very low (Rmin) and high (Rmax) [Ca2+], respectively; ratios at intermediate [Ca2+] were obtained by injection of solutions with different EGTA/Ca(2+)-EGTA proportions. This calibration gave an intracellular Ca2+ dissociation constant of indo-1 of 311 nM and a [Ca2+]i at rest of 52 +/- 4 nM (mean +/- SE; n = 15). Indo-1 records during twitches were compared with records obtained with the much faster indicator mag-indo-1. This analysis suggests a Ca2+ dissociation rate of indo-1 of 52 s-1 (22 degrees C). This makes indo-1 less suitable for measurements of [Ca2+]i during twitches, whereas it is fast enough to follow most aspects of [Ca2+]i during tetani, including the relaxation phase.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP132299219	Mag-Indo 1		132299-21-9	Price

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