Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria
Published: April 30, 2023
Abstract
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 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. Prepare 100 mL of KCl Buffer: 125 mM KCl, 20 mM HEPES, 1 mM…
Declarações
The authors have nothing to disclose.
Materials
| 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 |
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Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria. J. Vis. Exp. (Pending Publication), e21240, doi: (2023).