Determination of the Calcium Retention Capacity of Isolated Mitochondria

Abstract

Source: Li, W., et. al. Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay. J. Vis. Exp. (2018)

This video demonstrates the procedure to assess mitochondrial calcium retention capacity by monitoring real-time changes in fluorescence as mitochondria uptake calcium. This assay is useful for studying mitochondrial function and calcium-related processes, including apoptosis and neurodegenerative diseases.

Protocol

1. Determination of Mitochondrial Ca²⁺ Retention Capacity

1. Prepare the KCl media (125 mM KCl (potassium chloride), 2 mM K₂HPO₄ (dipotassium phosphate), 1 mM MgCl₂ (magnesium chloride), 20 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), 5 mM glutamate, 5 mM malate, and 2 μM rotenone, pH 7.4) 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 (calcium chloride) solution (20 mM CaCl₂, 127 mM KCl, 1 mM MgCl_2, 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 (mitochondrial permeability transition pore) opens, the fluorescence increases with time.
6. Interpret the total amount of Ca²⁺ added until the mPTP opens as the Ca²⁺ retention capacity.

Results

Figure 1: The software settings for mitochondrial Ca²⁺ retention capacity assay.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Calcium green-5N	Life Technologies	c-3737	Calcium binding green fluorescent dye
Glutamate	Sigma-Aldrich	RES5063G-A7
Malate	Sigma-Aldrich	46940-U
Rotenone	Sigma-Aldrich	R8875
HEPES	Sigma-Aldrich	H3375
Magnesium chloride	Sigma-Aldrich	457
Dipotassium phosphate	Sigma-Aldrich	V900050
KCl	Sigma-Aldrich	P9541
Calcium chloride	Sigma-Aldrich	V900266