hESC cultures are depleted of mtDNA according to the protocol of King and Attardi (1996).
1. Culture the hESCs in mtDNA depletion media for 5 d before treatment and change the media every day.
2. After 5 d, the hESCs are then grown in the presence of 50 ng/mL ethidium bromide for 2-4 wk to isolate cells completely lacking mtDNA. Again the media is changed every day and supplemented with fresh ethidium bromide.
3. After culture is depleted, a batch of cells is cultured in mtDNA depletion media. Transfer to aerobic media will test for depletion as cells devoid of mtDNA cannot respire aerobically.
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