Tmrm assay kit

TMRM assay was performed using TMRM Assay Kit (Abcam) following manufacturer’s protocol and visualized by using Keyence all-in-one florescence microscopy BZ-X710 (Ex/Em = 548/573). Fluorescence intensity in each image were measured by using Fuji software, subtracted to background signal, and normalized to total cell number in the image. Fluorescence intensity value in each well is the average of normalized background-subtracted florescence intensity of 3 random areas captured from each well.

For surface staining, cells were first incubated with Fc Receptor Blocker (CD16/32), followed by fluorochrome-labelled Abs of surface markers. For intracellular cytokine staining, Brefeldin A (Biolegend) was added for last 6 h of culture. For analysis of virus-specific CD4 and CD8 T-cell responses, cells were incubated with viral peptides GP33 (5 μg/ml) and GP61 (5 μg/ml) for 5 h in the presence of Brefeldin A. After incubation, cells were stained for surface markers first at 4°C for 30 min in the dark, followed by intracellular staining using IC Fixation Buffer (Thermo Fisher Scientific). For Ki-67 staining, the Foxp3/Transcription Factor Staining Buffer Set was used. The phosflow experiments were performed according to the protocol of BD Biosciences. Briefly, cells were stimulated with cytokines for the indicated times, followed by immediate fixation using a pre-warmed Cytofix Fixation Buffer at 37°C for 12 min. The cells were permeabilized using chilled Perm Buffer III for 1 h on ice and then were washed and stained with phosflow Abs. To measure the mitochondrial membrane potential, TMRM assay kit was used (Abcam). Samples were processed on an LSRII FACS Fortessa (BD Bioscience, San Jose, CA) and analysed using FlowJo X software (Tree Star, Ashland, OR).