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.
1. Reagents and Solutions
2. Isolation of Cardiac Mitochondria
3. Plate Reader-based Measurement of Mitochondrial Calcium Uptake
NOTE: Here, it is described the protocol for analyzing mitochondrial Ca2+ uptake using a multimode plate reader fitted with injectors. Any plate reader with the capability of reading calcium green-5N fluorescence (excitation/emission of 506/532 nm) in a kinetic mode with automated reagent injectors to keep the reaction protected from light can be used.
The authors have nothing to disclose.
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 |