2.1 Isolation of mitochondria from cell lines
Mitochondrial isolation was performed following a previously described
protocol.[17] Briefly, cells from two 150-mm
dishes were prepared in a homogenizing buffer. Subtilisin A solution
(200 µL, 4 mg/mL) was added to the buffer, and the solution was drawn
into a 22G syringe 20 times while being incubated in ice. The solution
was then centrifuged at 9,000 × g for 10 min at 4 °C. The pellet
was collected in 500 µL ice-cold respiration buffer, and used for
measuring ATP contents and MitoRAISE using the ATPlite Luminescence
Assay System (PerkinElmer,Waltham, MA, USA). The isolated mitochondria
was quantified using a Pierce BCA protein assay kit (Thermofisher
Scientific, Waltham, MA, USA).