Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Summary

Here, we present two protocols for the measurement of mitochondrial Ca²⁺ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca²⁺ import assay using the Ca²⁺ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca²⁺ dye Rhod-2/AM.

Abstract

Ca²⁺ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca²⁺ from mitochondria is important for determining the role of mitochondrial Ca²⁺ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca²⁺ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca²⁺ uptake using the Ca²⁺ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca²⁺ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca²⁺ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca²⁺ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca²⁺ in permeabilized cells using the Ca²⁺ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca²⁺. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca²⁺ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca²⁺ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Introduction

Mitochondria are critical sites of intracellular Ca²⁺ storage and signaling. Decades of research have demonstrated that mitochondria have the ability to import and sequester Ca^{2+ 1,2}. Mitochondria, however, are not merely passive sites of Ca²⁺ storage. Ca²⁺ at the mitochondrial compartment performs fundamental signaling functions including regulation of metabolic output and activation of mitochondrial-mediated cell death pathways, which has been reviewed previously³. For metabolic regulation, Ca²⁺ enhances the activity of three matr....

Protocol

All methods described in this protocol have been approved by the Institutional Animal Care and Use Committee of Emory University.

NOTE: The first part is the experimental procedure for measuring mitochondrial Ca²⁺ influx in isolated cardiac mitochondria using a plate reader.

1. Reagents and Solutions

1. 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 thro....

Results

Figure 1 shows mitochondrial Ca²⁺ uptake measurements in isolated cardiac mitochondria using the plate reader-based platform and the Ca²⁺ dye calcium green-5N. Under control conditions (Figure 1A), cardiac mitochondria were suspended in KCl buffer containing calcium green-5N and then challenged with sequential pulses of CaCl₂ (5 μL of a 0.6 mM CaCl₂ solution) added at the 30 s, 150 s, 3.......

Discussion

Here, we describe two different approaches to measure mitochondrial Ca²⁺ influx. The plate reader-based calcium green-5N method monitors extramitochondrial Ca²⁺ levels and is a Ca²⁺ uptake assay that is well suited for measurements in isolated mitochondria. While we have shown representative results from isolated murine cardiac mitochondria, this assay can be readily adapted for mitochondria isolated from tissues with high mitochondrial abundance including the liver, skeletal muscle, and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Olympus FV1000 Laser Scanning confocal microscope	Olympus	FV1000
Synergy Neo2 Multimode microplate reader with injectors	Biotek
Tissue Homogenizer	Kimble	886000-0022
22 x 22 mm coverslips	Corning	2850-22
96 well plate	Corning	3628
6 well plate	Corning	3506
Calcium Green-5N	Invitrogen	C3737
MitoTracker green FM	Invitrogen	M7514
Rhod-2, AM	Invitrogen	R1244
DMSO	Invitrogen	D12345
Pluronic F-127	Invitrogen	P3000MP
D-Mannitol	Sigma	M9546
Sucrose	EMD Millipore	8510
HEPES	Sigma	H3375
EGTA	Sigma	E8145
Potassium chloride	Fisher	BP366-500
Potassium phosphate monobasic	Sigma	P0662
Magnesium chloride	Sigma	M2670
Sodium pyruvate	Sigma	P2256
L-malic acid	Sigma	M1125
Calcium chloride	Sigma	C4901
Potassium acetate	Fisher	BP364-500
Adenosine 5′-triphosphate magnesium salt	Sigma	A9187
Phosphocreatine disodium salt	Sigma	P7936
Saponin	Sigma	S7900
Ru360	Calbiochem	557440