Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria

Published: April 30, 2023

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 plate reader-based assay to measure mitochondrial calcium uptake in isolated mitochondria. The assay analyzes the fluorescence signal of a calcium-sensitive fluorescent dye to study the kinetics of mitochondrial calcium uptake and calcium overload.

Protocol

1. Reagents and Solutions Make 500 mL of MS-EGTA buffer for mitochondrial isolation: 225 mM mannitol, 75 mM sucrose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), pH adjusted to 7.4 with KOH. Sterilize it through a 0.22 μm filter and store it at 4 °C. Ensure that the MS-EGTA buffer is pre-chilled to 4 °C before use. Prepare 100 mL of KCl Buffer: 125 mM KCl, 20 mM HEPES, 1 mM…

Divulgaciones

The authors have nothing to disclose.

Materials

Synergy Neo2 Multimode microplate reader with injectors Biotek
Tissue Homogenizer Kimble 886000-0022
96-well plate Corning 3628
Calcium Green-5N Invitrogen C3737
DMSO Invitrogen D12345
HEPES Sigma H3375
EGTA Sigma E8145
Potassium chloride Fisher BP366-500
Magnesium chloride Sigma M2670
Sodium pyruvate Sigma P2256
L-malic acid Sigma M1125
Calcium chloride Sigma C4901
Potassium phosphate monobasic Sigma P0662

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Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria. J. Vis. Exp. (Pending Publication), e21240, doi: (2023).

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