Mitoview green mvg

After culture, CD4⁺ T cells (0.2 × 10⁶) were stained with metabolic probes diluted in T-cell culture media for 20 min at 37° celsius. Cells were then washed twice in staining buffer (PBS/2% FCS) and resuspended before analysis. Metabolic probes used included MitoSpy Orange (MSO) (25 nM; BioLegend, Cat# 424803), and MitoView Green (MVG) (50 nM; Biotium, Cat# 70054). For analysis of c-Myc expression and p70S6K phosphorylation, cells (0.2 × 10⁶) were first fixed in FoxP3 fixation/permeabilization solution (eBioscience, Cat# 005523–00) for 20 min at 37° celsius, washed in FoxP3 permeabilization buffer and incubated with anti-c-Myc (Cell Signalling Technology, Cat# 5605T) or anti-phospho p70S6K (Thr421/ Ser424) (Cell Signalling Technology, Cat# 9204S) for 30 min at 4° celsius. After washing, cells were then incubated with secondary antibody anti-Rabbit IgG (H + L), AF555 (Invitrogen, Cat# A-21428) for 20 min at 4° celsius, followed by a final wash. Samples were run on the BD LSRFortessa X-20, data collected using BD FACSDiva Software, and analysed using FlowJo version 10.7.1 (BD Biosciences).