Mitochondria purification

HEK293T cells were trypsinized (0.05% Trypsin 0h 5m 0s ) and collected by centrifugation (500g 0h 5m 0s).

Cells were washed twice with NKM buffer (1 mM Tris HCl, pH7.3, 0.13 M NaCl, 5 mM KCl, and 7.5 mM MgCl2), and resuspended in six packed cell volumes of homogenization buffer (10 mM Tris pH 7.4, 10 mM KCl, and 0.15 mM MgCl2).

Cells were homogenized by 10 passages through a 22G needle.

Cell homogenates were mixed gently with the same volume of 2.3 M sucrose solution and centrifuged at 1200×g for 0h 5m 0s 4°C to remove unbroken cells and large cell debris.

The recovered supernatant fractions were centrifuged at 7000×g for 0h 10m 0s 4°C .

Mitochondria enriched in the pellet fraction were resuspended in three packed cell volumes of Mitochondria Suspension Buffer (10 mM Tris, pH 7.3, 0.15 mM MgCl2, and 0.25 mM

sucrose).