ATP assay

The ATP bioluminescence assay was used to quantify ATP levels. TIL and PBL samples were labeled with CD8 microbeads (Miltenyi Biotec; #130-045-201, MACS) and then positively selected for CD8 T cells by passing them through the LS column (Miltenyi Biotec; #130-042-401, MACS) according to the manufacturer’s instructions. ATP levels of the positive-selection CD8 T cells were determined using a Luminescent ATP Detection Assay kit (Abcam; ab113849) following the manufacturer’s instructions. Briefly, CD8 T cells were incubated with detergent for 5 min, followed by incubation with substrate solution for 10 min at room temperature in the dark with agitation. Then, luminescence was quantified on a microplate reader (Molecular Devices; SpectraMax M2). The luminescence values of ATP standards were determined in a similar manner. ATP concentration was calculated according to an ATP-standard curve.