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 Ca²⁺ also take part in the cytosolic calcium signals and activate the mitochondrial apoptotic mechanism by inducing the opening of the mPTP in the inner mitochondrial membrane². It has been widely recognized that abnormal elevation of intracellular Ca²⁺ can induce mitochondrial massive swelling by opening the mPTP³. Thus, this protocol aims to assess the mitochondrial function by quantifying the mitochondrial Ca²⁺ retention capacity and Ca²⁺-trigged mitochondrial swelling.

Ca²⁺ retention capacity is the measurement of the ability of mitochondria to uptake cytosolic calcium. Mitochondria can buffer the cytosolic free calcium and regulate calcium-dependent cellular processes by calcium uptake in the form of inactive precipitates. Impaired Ca²⁺ retention capacity occurs in stress phenomena associated with energy limitation and even neurodegenerative diseases^4,5. Isolated mitochondria or digitonin-permeabilized cells can be used to identify the Ca²⁺ retention capacity, and the elevated ability to accumulate Ca²⁺ by isolated mitochondria with the additonal glutamate and malate is not effected by the mitochondrial isolation procedure⁶. A titrated amount of digitonin should be used to permeabilize the plasma membranes of different cell types. The hexapotassium salt of Ca²⁺-binding green fluorescent dye, a Ca²⁺-sensitive cell-impermeant visible light-excitable indicator, has been widely used⁷. Ca²⁺-binding green fluorescent dye is a low-affinity indicator and used to show that intracellular free Ca²⁺ concentrations continue to rise during prolonged (5 min) stimulations⁸. This method was less sensitive than the radioactive filter technique but was greatly simplified. The additional Ca²⁺ elevates calcium green fluorescence and the Ca²⁺ uptake by mitochondria returns the fluorescence to baseline. Sequential additions of Ca²⁺ were made until the mitochondria failed to uptake extramitochondrial Ca^{2+ 9}. A fluorescence microplate reader can be used to continuously report the calcium green fluorescence.

After Ca²⁺ accumulation, mitochondria depolarized, released Ca²⁺ into the medium, and began to swell. Ca²⁺-triggered mitochondria swelling is used to indicate the mPTP opening. Electron microscopy and the decrease in light absorbance at 540 nm can be used to measure the Ca²⁺-triggered mitochondrial swelling^10,11. Mitochondria volume can be directly determined by forward angle light scattering¹², where decreases in the absorbance reflect passive swelling of the mitochondrial matrix.

Here, we illustrate the methodology to examine the mitochondrial Ca²⁺ retention capacity and Ca²⁺-triggered mitochondrial swelling in isolated mitochondria from SH-SY5Y cells.

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.
3. Add new 1 mL of ice cold PBS into 10 cm cell culture dish and scrape the adherent cells into a new 1.5 mL tube. Centrifuge at 800 x g for 10 min at 4 °C.
4. During the centrifugation, prepare 5 mL mitochondrial isolation buffer (250 mM sucrose, 10 mM HEPES(4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA) supplemented with 50 μL of protease inhibitors stock solution (100x concentration; 1 EDTA-free tablet dissolved in 500 µL of ddH₂O).
5. Remove the supernatant and resuspend the pellet with 1 mL of mitochondrial isolation buffer. Incubate the 1.5 mL tube on ice for 30 min.
6. Lyse the suspended cells with a glass homogenizer (15 - 25 strokes) up and down manually on ice.
   NOTE: Make sure there are no bubbles when cells are homogenized. Count the intact cells and bared nuclei by microscope visually and confirm that >90% cell breakage has occurred.
7. Transfer the homogenate into a 1.5 mL tube and centrifuge at 1,000 x g for 5 min at 4 °C to remove the debris (nuclei and remaining intact cells).
8. Transfer the supernatant into a new 1.5 mL tube and centrifuge at 1,000 x g for 5 min 4 °C.
9. Transfer the supernatant to a new tube and centrifuge at 14,000 x g for 15 min 4 °C.
10. Remove the supernatant and resuspend the pellet containing mitochondria with 1 mL of mitochondrial isolation buffer.
11. Centrifuge the solution for 15 min at 14,000 x g 4 °C to precipitate the mitochondria.
12. The resulting mitochondria pellet must be kept on ice and resuspended in 50 - 100 μL KCl media (125 mM KCl, 2 mM K₂HPO₄, 1 mM MgCl₂, 20 mM HEPES, 5 mM glutamate, 5 mM malate, and 2 μM rotenone, pH 7.4) according to the pellet volume before any assay.
13. Use 5 μL of mitochondrial solution to measure the mitochondrial protein concentration by standard BCA assay, measuring OD562 using a plate reader¹³.

2. Determination of Mitochondrial Ca²⁺ Retention Capacity

1. Prepare the KCl media and add the Ca²⁺-binding green fluorescent dye to a final concentration of 0.5 μM before the experiment.
2. Transfer 1 mL of isolated mitochondria (0.4 mg/mL) in KCl media containing 0.5 μM Ca²⁺-binding green fluorescent dye into each 6-well plate well.
3. Incubate the mitochondria and the Ca²⁺-binding green fluorescent dye in the 6-well plate at room temperature protected from ambient light for 1 min. Add 4 μL aliquots of a 20 mM CaCl₂ solution (20 mM CaCl₂, 127 mM KCl, 1 mM MgCl₂, 20 mM HEPES, 5 mM glutamate, 5 mM malate, pH 7.4) to each 6-well plate well using the automatic dispense setting to introduce 200 nmol Ca²⁺/mg mitochondrial protein.
4. Use a fluorescence spectrometer to monitor the fluorescence changes every 3 s for 2 min with an excitation wavelength of 506 nm and an emission wavelength of 531 nm. The plate is equipped with shaking at 600 rpm for 3 s between readings controlled by software (Figure 1).
5. Add an additional 4 μL aliquot of 20 mM CaCl₂ solution to each well and monitor the fluorescence changes every 3 s for 2 min. Repeat this step until the fluorescence increases with time.
   NOTE: Mitochondria are challenged with the added Ca²⁺ indicated by the increased recording fluorescence. When the mPTP opens, the fluorescence increases with time.
6. Interpret the total amount of Ca²⁺ added until the mPTP opens as the Ca²⁺ retention capacity.

3. Determination of Ca ²⁺-induced Mitochondrial Swelling

1. Prepare the KCl media before the experiment.
   1. Transfer 1 mL of isolated mitochondria (0.4 mg/mL) in KCl media into each 6-well plate well.
2. Add 10 μL of 20 mM CaCl₂ aqueous solution (500 nmol/mg mitochondrial protein) into mitochondrial protein to trigger mitochondrial swelling.
3. Record the decrease in absorbance at 540 nm every 3 s on a microplate reader controlled by software for 10 min (Figure 2). The fluorescence changes are caused by an influx of solutes across the mitochondrial inner membrane.
   NOTE: The decreased absorbance at 540 nm corresponds to the swollen mitochondria. If the reader does not observe a decrease in absorbance, a longer reading interval or reading time can be adopted.

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