Facsaria fusion equipment

Cells were cultured in a humidified incubator at 37 °C with a controlled atmosphere of ambient air 10% CO₂. Murine colorectal carcinoma cell line CT26 (ATCC, CRL-2638) or IF1-KO CT26 were grown in RPMI media supplemented with 10% fetal bovine serum (FBS, MilliporeSigma, F7524). Murine colorectal carcinoma cell line MC38 (Kerafast, ENH204-FP) or IF1-KO MC38 were grown in DMEM media supplemented with 10% fetal bovine serum (FBS, MilliporeSigma, F7524). The generation of stable IF1-KO cells was carried out by the CRISPR-Cas9 technique [12 (link)]. Cells were co-transfected with two plasmids (MLM3636-sgRNA-mIF1-1 and MLM3636-sgRNA-mIF1-2) that express two guides for Atp5if1 and a plasmid that contains the nuclease Cas9 and GFP (pSpCas9(BB)-2A-GFP, PX458) (Addgene, RRID: Addgene_48138). Transfection was carried out using Lipofectamine 3000 (Thermo Fisher, L3000015) following the manufacturer’s instructions. After 48 h, GFP⁺ cells were sorted in FACSAria Fusion equipment (BD Biosciences) from the Cytometry Facility (CBMSO, Spain), and they were expanded for future experiments. Cells transfected with a plasmid that express a scramble guide and the plasmid that contains the nuclease Cas9 and GFP were used as controls. The Atp5if1 CRISPR-Cas9 mediated gene knockout was checked by Western blot.

HPMC suspensions were preincubated with human Fc Receptors block (#564220 from BD Biosciences) to reduce nonspecific binding. Cells were stained with podoplanin-BV421 and CD45-FITC conjugated antibodies. Antibodies were omitted in negative controls. Flow cytometry data acquisition was performed on a FACSAria Fusion equipment (BD Biosciences). FlowJo software (Tree Star Inc., Ashland, OR) was used for data analysis.