Calcium Influx Assay to Measure Mitochondrial Calcium Uptake in Cultured Cells
Transcrição
The mitochondrial uptake of cytosolic calcium ions via its inner membrane mitochondrial uniporter complex is crucial for its functioning.
To measure the mitochondrial calcium influx, begin with a multi-well plate containing an ECM-coated coverslip at the bottom of its well. Plate the fibroblast suspension onto the coverslip, and allow the cells to adhere to the ECM.
Add a dye mix containing an inactive, esterified red-fluorescent calcium-sensitive dye and a green-fluorescent mitochondria-selective dye. Incubate to allow the dye molecules to enter the cell cytosol.
The mitochondria-sensitive dye selectively diffuses through its membrane and localizes in the mitochondrial matrix. The calcium-sensitive dye spreads throughout the mitochondria and extra-mitochondrial spaces. Inside the mitochondrial lumen, endogenous esterases cleave the ester moiety from the red dye, releasing a membrane-impermeable red fluorophore that remains trapped inside the mitochondria.
Add a surfactant-containing permeabilization solution. At low concentrations, surfactant molecules restrictively dissolve the cells' plasma membrane cholesterol, leaving their mitochondria intact and creating spaces through which calcium-sensitive dye molecules leak out of the cytoplasm. This selectively retains the calcium-sensitive red fluorophores and mitochondria-specific green fluorophores in the mitochondria.
Transfer the coverslip to an imaging chamber fixed on a confocal microscope, and add a calcium-containing buffer. Within the permeabilized cells, locate regions displaying co-localized red and green fluorescence, indicating mitochondria-localized calcium. The red fluorescence gradually augments, depicting progressive mitochondrial calcium uptake.
