Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

Summary

This protocol aims to describe a method to examine the Ca²⁺ retention capacity and Ca²⁺- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

Abstract

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca²⁺ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca²⁺ concentration. Ca²⁺ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca²⁺ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca²⁺-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca²⁺ retention capacity and Ca²⁺-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca²⁺ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca²⁺-related mitochondrial functions.

Introduction

Mitochondria are the main cellular organs to produce nearly 95% of the ATP used in the mammalian cells by oxidative phosphorylation. It is reported that the sequestered micromolar concentration of Ca²⁺ by mitochondria, the presence of ADP, and inorganic phosphate can be used to phosphorylate ADP to synthesis ATP¹. When the concentration of cytosolic Ca²⁺ goes above a threshold, mitochondria can uptake Ca²⁺ rapidly and efflux it slowly. Thus, the functioning mitochondria can influx the increased cytosolic Ca²⁺. Irrelevant to the participation in the oxidative phosphorylation, the mitochondrial Ca^2....

Protocol

1. Isolation of Mitochondria

NOTE: All solutions and equipment should be precooled to 0 - 4 °C and kept on ice.

1. Seed about 3 x 10⁶ SH-SY5Y cells per 10 cm cell culture dish. Culture cells in high-glucose Dulbecco's modified Eagle medium with 10% fetal bovine serum, penicillin (100 U/mL)-streptomycin (100 µg/mL). Maintain at 37 °C in an incubator containing 5% CO₂ overnight.
   NOTE: At least 1 - 2 x 10⁷ SH-SY5Y cells are needed for each isolation assay.
2. Remove the medium and wash the cells with about 1 mL of ice cold PBS in a 10 cm cell culture dish 3 times.
....

Results

Representative Results of Ca²⁺ Retention Capacity:
Results were expressed as fluorescence values. With the additional pulses of Ca²⁺ (200 nmols/mg mitochondrial protein), the fluorescence increased by double above baseline with the mPTP opening. 5 μM Bongkrekate (BKA), an inhibitor of Ca²⁺-induced mPTP opening, or 1 μM atractyloside (ATR), an activator of Ca²⁺-induced mPTP opening, were added in isolated mitochondria. The amount of .......

Discussion

Here, we described a simple and effective protocol for the mitochondrial Ca²⁺ retention capacity assay and Ca²⁺-triggered mitochondrial swelling assay.

For the mitochondrial isolation, make sure that all the materials and tubes are on ice, especially when homogenizing the cells. 0.25% trypsin-EDTA can also be used to detach cells from the dish for 5 min at 37 °C. After 15 - 25 strokes, it is necessary to observe the intact cell membrane under the microscope and avoid.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SH-SY5Y cell line	ATCC (The Global Bioresource Center)	ATCC Number: CRL-2266
Calcium green-5N	Life Technologies	c-3737	Ca2+-binding green fluorescent dye
complete protease inhibitors	Roche Molecular Biochemicals	4693116001
glass homogenizer	Kimble Chase	9885303002
glutamate	Sigma-Aldrich	RES5063G-A7
malate	Sigma-Aldrich	46940-U
rotenone	Sigma-Aldrich	R8875
HEPES	Sigma-Aldrich	H3375
MgCl2	Sigma-Aldrich	00457
K2HPO4	Sigma-Aldrich	V900050
KCl	Sigma-Aldrich	P9541
CaCl2	Sigma-Aldrich	V900266
Varioskan flash instruments	Thermo Scientific	IC100E
Bongkrekate	Biovision	1820-100
atractyloside	Sigma-Aldrich	C4992
high-glucose Dulbecco’s modified Eagle medium	Hyclone	SH30022	4.5 g/L glucose, L-glutamine, without sodium pyruvate
fetal bovine serum	Hyclone	SV30087
penicillin	Sigma-Aldrich	P3032
streptomycin	Invitrogen	11860-038
BCA assay kit	Thermo Scientific	NCI3225CH
PBS	Hyclone	SH30256.01
10 cm cell culture dish	NEST	704001