Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Summary

Mitochondria can utilize the electrochemical potential across their inner membrane (Δ_Ψm) to sequester calcium (Ca²⁺), allowing them to shape cytosolic Ca²⁺ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca²⁺ uptake and ΔΨ_m in live cells using fluorescent dyes and confocal microscopy.

Abstract

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca²⁺) buffers to tightly regulate intracellular Ca²⁺ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (ΔΨ_m) to sequester Ca²⁺. The influx of Ca²⁺ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains ΔΨ_m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.

We describe here a method for simultaneously measuring mitochondria Ca²⁺ uptake and ΔΨ_m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca²⁺ can be measured using the fluorescent Ca²⁺ indicator Fluo-4, AM, with measurement of ΔΨ_m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca²⁺ and ΔΨ_m simultaneously. Using the sequential addition of Ca²⁺ aliquots, mitochondrial Ca²⁺ uptake can be monitored, and the concentration at which Ca²⁺ induces mitochondrial membrane permeability transition and the loss of ΔΨ_m determined.

Introduction

Mitochondria play an important role in regulating intracellular Ca²⁺ concentration by acting as local Ca²⁺ buffers¹. Ca²⁺ enters the mitochondria via the Ca²⁺ uniporter, a process driven by the electrochemical gradient that exists across the mitochondrial inner membrane (ΔΨ_m)². Once inside the mitochondrial matrix, Ca²⁺ can activate oxidative phosphorylation by stimulating three rate-limiting dehydrogenases of the citric acid cycle³. This stimulation maintains ΔΨ_m, which is temporarily dissipated as th....

Protocol

1. Preparation of Cells

1. Grow cells on 10 cm cell culture dishes or 75 cm² flasks in culture media [10 ml Dulbecco's Modified Eagles Medium (DMEM) supplemented with 5% (v/v) fetal bovine serum (FBS) and 1x penicillin/streptomycin (p/s)] at 37 °C/5% CO₂.
2. To harvest cells, remove media by aspiration, then wash with 5 ml 1x phosphate buffered saline (1x PBS). Remove 1x PBS by aspiration, then add 1.5 ml 0.25% (w/v) Trypsin/0.25% (w/v) ethylenediaminetetraacetic acid (EDTA) and incubate at 37 °C/5% CO₂ for 2 min. Tap dish or flask gently to remove cells, then resuspend in 5 ml culture media.
3. Cou....

Results

We have used this protocol to examine the effects of an MT-ND5 mutation on the ability of 143B cell mitochondria to buffer increases in calcium¹². In the example shown here, control 143B cells were loaded with TMRM and Fluo-4, AM before permeabilization with digitonin. After 5 min of imaging, eight sequential additions of a 1:100 dilution of 40 mM exogenous CaCl₂ were made, with the final free Ca²⁺ ion concentration [Ca²⁺] .......

Discussion

Calcium plays a critical role in many cell processes, including muscle contraction, neuronal signaling and cell proliferation¹³. Increases in cell calcium concentrations are often associated with energy demand, with calcium able to directly stimulate mitochondrial oxidative phosphorylation to raise ATP generation³. It is therefore essential that we have the ability to effectively monitor mitochondrial calcium accumulation and to be able to compare how this function is affec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Modified Eagle Medium (DMEM)	ThermoFisher	10566016
fetal bovine serum (FBS)	ThermoFisher	16000044
1x phosphate buffered saline (PBS)	ThermoFisher	10010023
100x penicillin/streptomycin (p/s)	ThermoFisher	15140122
0.25% Trypsin/0.25% EDTA	ThermoFisher	25200056
8-well chambered coverslip	ibidi	80826
NaCl	Sigma-Aldrich	793566
KCl	Sigma-Aldrich	P9541
MgSO4	Sigma-Aldrich	746452
KH2PO4	Sigma-Aldrich	795488
D-glucose	Sigma-Aldrich	G8270
CaCl2	Sigma-Aldrich	746495
HEPES	Sigma-Aldrich	H3375
MgCl2	Sigma-Aldrich	M2670
EGTA	Sigma-Aldrich	E4378
HEDTA	Sigma-Aldrich	H8126
malate	Sigma-Aldrich	M1000
glutamate	Sigma-Aldrich	G1626
ADP	Sigma-Aldrich	A5285
Ca2+ free Hank’s buffered salt solution (HBSS)	ThermoFisher	14175-095
tetramethylrhodamine, methyl ester, perchlorate (TMRM)	ThermoFisher	T668
Verapamil	Sigma-Aldrich	V4629
Fluo-4 acetoxymethyl ester (Fluo-4, AM)	ThermoFisher	F14201
dimethyl sulfoxide (DMSO)	ThermoFisher	D12345
carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)	Sigma-Aldrich	C2920
digitonin	Sigma-Aldrich	D141
thapsigargin	Sigma-Aldrich	T9033
Pluronic F-127	ThermoFisher	P3000MP
hemacytometer	VWR	631-0925
10 cm cell culture dishes	Corning	COR430167
75 cm2 cell culture flasks	Corning	COR430641