Cells were seeded in 12-well plates and transfected for 24 h as described above. Afterwards, cells were incubated with 20 nM tetramethylrhodamine methyl ester (TMRM; Molecular Probes, Eugene, OR, USA) for 15 min at 37°C, harvested and analyzed by flow cytometry on a BD-FACS canto Flow Cytometer8 (link),10 (link). As positive control, mitochondrial depolarization was elicited using 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP; Sigma Aldrich, St. Louis, MO, USA) for 30 min at 37°C.
Borgna V., Lobos-González L., Guevara F., Landerer E., Bendek M., Ávila R., Silva V., Villota C., Araya M., Rivas A., López C., Socias T., Castillo J., Alarcón L., Burzio L.O., Burzio V.A, & Villegas J. (2020). Targeting antisense mitochondrial noncoding RNAs induces bladder cancer cell death and inhibition of tumor growth through reduction of survival and invasion factors. Journal of Cancer, 11(7), 1780-1791.
Other organizations :
Fundación Ciencia and Vida, Universidad de Santiago de Chile, Hospital Barros Luco-Trudeau, Universidad del Desarrollo, Clínica Alemana, San Sebastián University, Universidad Bernardo O'Higgins, Universidad Andrés Bello
Mitochondrial membrane potential measured by TMRM fluorescence
control variables
Cell seeding in 12-well plates
TMRM staining concentration (20 nM)
TMRM incubation time (15 min)
TMRM incubation temperature (37°C)
controls
Positive, Mitochondrial depolarization induced by 10 µM CCCP for 30 min at 37°C
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