Is anybody successful in depletion of mitochondrial DNA in MCF10A cells by ethidium bromide treatment? Cells cultured in DMEM (4.5 g glucose/L) supplemented by horse serum, EGF, cholera toxin, human insulin, penicilin/streptomycin (acording to http://muthuswamylab.cshl.edu/protocols/culturing_MCF-10A.pdf) and enriched by uridine, pyruvate and non-essential amino acids (Gly, Ala, Asp, Asn, Glu, Pro, Ser) when treated, EtBr at final concentration 0,2 ug/mL. Cells don’t survive a week, neather in 0,1 ug/mL EtBr nor in recommended DMEM/F12. Any advice?
Jan, we tried to generate Rho(0) cells from MCF-10A with no success. Unlike many cancer cell lines, these cells can't survive even very low concentrations of the EtBr. If you can't replace them to the different cell line, my suggestion is to try a mitochondrially-targeted restriction endonuclease approach (see the attached paper).
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