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.
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