Nature research paper: Spatial mapping of mitochondrial networks and bioenergetics in lung cancer
, 1 mM EGTA, 2 mM HEPES pH 7.4) with protease inhibitor cocktail . Homogenates were centrifuged at 1,000for 10 min at 4 °C and supernatant was collected. Protein concentrations were determined by BCA assay kit . Homogenates were loaded into a Seahorse XF96 microplate in MAS buffer and centrifuged at 2,000for 5 min at 4 °C. After centrifugation, the volume was increased to 150 µl by adding 130 µl MAS containing cytochrome.
. Homogenates were stained with 500 nM for 10 min followed by two wash steps to remove the dye . fluorescence was read on a Tecan Spark plate reader .Cells were seeded into a Seahorse XF96 microplate before the assay and maintained in a tissue culture incubator to reach 90–100% density before running the Seahorse assay. To measure complex I and complex II respiratory capacity, cells were permeabilized with 4 nM XF plasma membrane permeabilizer .
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