Resuspend mitochondria in one-third of the packed cell volume with mitochondrial lysis buffer. Put the suspension into a glass homogenizer and homogenize with 10 strokes using a tight pestle. Add Tween 20 and KCl to final concentrations of 0.5% and 0.5 M, respectively.
Incubate the mixture on ice for 5 min. Repeat the homogenization 10 times.
Spin the final mitochondrial lysate at 100,000 xg in an ultracentrifuge using a TY65 Beckman rotor at 4°C, for 1 h.
Carefully collect the clear supernatant, avoiding the fluffy layer over the pellet, to yield the final S-100 fraction.
Freeze in aliquots, in liquid nitrogen, and store at -80°C.
This is a modified protocol taken from: Micol V, Fernandez-Silva P and Attardi G (1996). Mitochondrial Biogenesis and Genetics Part B. Methods of Enzymology, Volume 264, p 3–621.
For convenience please consider our Mitochondria Isolation Kit for Cultured Cells (ab110171).