Genjet dna in vitro transfection reagent version 2

Heteroplasmic cybrids harboring the m.8344A>G mtDNA, generated in our laboratory as previously described (Hashimoto et al, 2015), were transfected with 30 μg of plasmid using GenJet DNA In Vitro Transfection Reagent version II (SL100489, SignaGen Laboratories) in a T‐75 flask, following the manufacturer's instructions. The cells were collected either 24 h, 48, or 72 h post‐transfection for cell sorting analysis. Sorting was performed in a FACS Aria IIU by gating on single‐cell fluorescence using a 561‐nm laser and 600LP, 610/20 filter set for mCherry; and a 488‐nm laser and 505LP, 530/30 filter set for eGFP. For some experiments, we also used a Beckman Coulter MoFlo Astrios EQ using a 100 μm nozzle at 25psi. Sorted cells were collected into separate tubes, one of them containing the sorted fluorescent cells either termed “GFP++” and “GFP+++” for some of the sortings, in the case of the mitoTev‐TALEs; or “Yellow” when co‐transfected with the two monomers containing mCherry and eGFP, in the case of the mitoTALEN, as previously described (Hashimoto et al, 2015); we also isolated the untransfected sorted population of cells lacking fluorescence which were termed “Black” for the mitoTALENs and termed “GFP−+” for the mitoTev‐TALE. The controls were also “sorted”, in order to mimic the same conditions for the three or four cell populations.