Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria

Samenvatting

Source: Maxwell, J.T., et al., Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells. J. Vis. Exp. (2018).

This video describes a plate reader-based assay to measure mitochondrial calcium uptake in isolated mitochondria. The assay analyzes the fluorescence signal of a calcium-sensitive fluorescent dye to study the kinetics of mitochondrial calcium uptake and calcium overload.

Protocol

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 through a 0.22 μm filter and store it at 4 °C. Ensure that the MS-EGTA buffer is pre-chilled to 4 °C before use.
2. Prepare 100 mL of KCl Buffer: 125 mM KCl, 20 mM HEPES, 1 mM KH₂PO₄, 2 mM MgCl₂, 40 μM EGTA and pH adjusted to 7.2 with KOH. Store it at room temperature.
3. Prepare 1 mM ca....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Synergy Neo2 Multimode microplate reader with injectors	Biotek
Tissue Homogenizer	Kimble	886000-0022
96-well plate	Corning	3628
Calcium Green-5N	Invitrogen	C3737
DMSO	Invitrogen	D12345
HEPES	Sigma	H3375
EGTA	Sigma	E8145
Potassium chloride	Fisher	BP366-500
Magnesium chloride	Sigma	M2670
Sodium pyruvate	Sigma	P2256
L-malic acid	Sigma	M1125
Calcium chloride	Sigma	C4901
Potassium phosphate monobasic	Sigma	P0662