Summit acquisition software

Purified CD4⁺ T cell populations were obtained from spleen and lung using flow-cytometric cell sorting on a MoFlo® Astrios™ with Summit acquisition software (Beckman Coulter, Brea, CA, USA). Cells were cultured with and without PPD-T at 10 µg/ml with the addition of 1 µg/ml anti-CD28 for 3 days at 37 °C/5% CO₂, in co-culture with adherent spleen-derived APC from naïve congenic CD90.1 BALB/c mice. Briefly, spleens were processed as previously and incubated for 4 h/37 °C before removal of non-adherent cells by three aspirations of supplemented DMEM. Antigen and purified CD4⁺ T cells were immediately added to the culture. Any congenic T cells which had failed to be removed from the splenic APC co-culture were excluded from final flow-cytometric analysis through expression of CD90.1. Cell-surface staining was performed as previously with CD90.1-BV421, CD44-BV785, live/dead-Zombie Aqua (all BioLegend) and CD4-APC-H7 (BD Biosciences). After surface staining, cells were washed twice as previously and cold 70% ethanol added to the cell pellet while vortexing. Cells were incubated for 1 h/−20 °C, washed three times, stained for 30 min/room temperature with Ki67-PE (BioLegend) and washed twice before analysis, as previously.

HEK293T and HeLa cells were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol.
MELAS-iPSCs were cultured under feeder-free conditions in accordance with a previous report^{35 (link)} for transduction of mpTALEN plasmids. After removing feeder cells, iPSCs were dissociated into single cells by incubation with 0.5× TrypLE Select (Gibco) for about 4 min at 37 °C. Single iPSCs were reseeded on dishes coated with iMatrix-511 (Nippi, Japan) and cultured using StemFit AK02N medium (Ajinomoto). One or two days later, pCAGGS-EGFP (pCAGGS³⁶ vector provided by RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan) and L- and R-mpTALEN plasmids were introduced into iPSCs using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s protocol. One or two or three days later, cells were harvested and sorted using a Moflo Astrios (Beckman Coulter) with Summit acquisition software (Beckman Coulter). A sorting gate was established based on forward and side scatter as well as the level of EGFP expression after exclusion of dead cells and debris stained with propidium iodide (PI). EGFP-positive cells were directly sorted into primate embryonic stem cell medium (ReproCell) with 10 μM Y-27632 (Wako).

MELAS-iPSCs were cultured under feeder-free conditions for the transfection of the mpTALEN plasmids. Single iPSCs were reseeded on dishes coated with iMatrix-511 (Matrixome) and cultured in StemFit AK02N medium (Ajinomoto) or primate ESC medium (ReproCELL) without bFGF. One day later, pCAGGS-EGFP⁸ (link) and the mpTALEN plasmids (2 μg each) were introduced into iPSCs using Lipofectamine 3000 reagent (Invitrogen) according to the manufacturer’s protocol. Two days later, the cells were harvested and sorted using a MoFlo Astrios instrument (Beckman Coulter) with Summit acquisition software (Beckman Coulter) as previously described.⁸ (link) A sorting gate was established based on the forward and side scatters, as well as on the level of EGFP expression, after the exclusion of dead cells and debris, which were stained with PI. EGFP-positive and PI-negative cells were directly sorted into StemFit AK02N or primate ESC medium with 10 μM Y-27632.
HEK293T and HeLa cells were transfected using Lipofectamine 3000 reagent according to the manufacturer’s protocol.