Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells

Abstract

Source: Maxwell, J.T., et al., Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells. J. Vis. Exp. (2018).

This video describes a confocal microscopy imaging-based assay to measure mitochondrial calcium uptake in cultured cells. The assay uses the calcium-sensitive fluorescent dye Rhod-2/AM to measure the mitochondrial calcium in permeabilized cultured cells.

Protocol

1. Reagents and Solutions NOTE: The second part is experimental procedure for confocal imaging of mitochondrial Ca2+ in cultured cells Make Tyrode's solution (130 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.2 with KOH). Store it at 4 °C. Warm it to room temperature before use. Prepare Wash Solution (100 mM potassium acetate, 15 mM KCl, 5 mM KH2PO4, 5 mM Mg-…

Materials

Olympus FV1000 Laser Scanning confocal microscope	Olympus	FV1000
22 x 22 mm coverslips	Corning	2850-22
6-well plate	Corning	3506
MitoTracker green FM	Invitrogen	M7514
Rhod-2, AM	Invitrogen	R1244
DMSO	Invitrogen	D12345
Pluronic F-127	Invitrogen	P3000MP
HEPES	Sigma	H3375
EGTA	Sigma	E8145
Potassium chloride	Fisher	BP366-500
Potassium phosphate monobasic	Sigma	P0662
Magnesium chloride	Sigma	M2670
Calcium chloride	Sigma	C4901
Potassium acetate	Fisher	BP364-500
Adenosine 5′-triphosphate magnesium salt	Sigma	A9187
Phosphocreatine disodium salt	Sigma	P7936
Saponin	Sigma	S7900