Fortessa flow cytometer

As previously described, total live mononuclear cells were isolated from fresh tumor specimens (Supplementary Table 7, 8)^{51 (link)}. Tumor-derived single cells suspensions were split into halves. One half was depleted of B cells using EasySep™ HLA Chimerism Whole Blood B Cell Positive Selection Kit (EasySep) according to the manufacturer’s instructions. The second half was subjected to the same procedures without addition of microbeads. After magnetic separation, both cell suspensions were cultured in RPMI 1640 supplemented 10% inactivated FCS, l-glutamine and penicillin–streptomycin (Invitrogen) in 24-well plates for 1 day without any additional stimuli. The purity and cell yield of the magnetic separation was assessed using panel of fluorescent primary antibodies or appropriate isotype controls for 20 min at 4 °C in the dark, followed by washing and acquisition on a Fortessa flow cytometer (BD Biosciences). The phenotype of CD8⁺ T cells was asseseed at day 2 using panel of fluorescent extracelluar and intracelluar primary antibodies or appropriate isotype controls for 20 min at 4 °C in the dark, followed by washing and acquisition on a Fortessa flow cytometer (BD Biosciences) (Supplementary Table 10). Flow cytometry data were analyzed with the FlowJo software (TreeStar) (Supplementary Fig. 8).

For TMRM staining, mitochondria were harvested according to protocol detailed here, resuspended in DMEM supplemented with 1 mM l-pyruvate and 200 nM TMRM and incubated for 30 min at 37 °C and 5% CO₂. After staining, mitochondria were washed twice in PBS, before resuspension in PBS, followed by flow cytometric analysis using a Fortessa flow cytometer (BD Biosciences). For MitoView staining, cells were harvested by trypsinisation, and centrifugation, and washed twice in warmed PBS. Cells resuspended in warmed PBS, or warmed PBS containing 200 nM MitoView (green). Fluorescence intensity was measured using a FACSCalibur flow cytometer (BD Biosciences). For 2-NBDG uptake assay, cells in suspension were incubated in warmed (37 °C) PBS or warmed PBS containing 200 μM 2-NBDG for 20 min. Cells were then washed three times in warmed PBS followed by flow cytometric analysis using a Fortessa flow cytometer (BD Biosciences).

IκBα degradation assay: Cryopreserved PBMCs were thawed in a 37 °C water bath and suspended RPMI1640 + 10% human AB serum and cultured overnight. Cells washed and incubated at 10⁶ cells per ml in 500 µl media per well, supplemented where appropriate with TNF-α (10 ng/ml) or D18 (1 µg/ml) for 15 min at 37 °C/5% CO₂. Cells were washed and stained with surface markers and fixed/permeabilised according to the manufacturer’s instruction. Then cells were stained with PE-conjugated control antibody or IκBα for 15 min at room temperature^{16 (link)}. Cells were washed, fixed and analysed promptly using a BD Fortessa flow cytometer.
NFκB2 and Ki67 assay: PBMC were treated with TNF-α (10 ng/ml), or D18 (1 µg/ml), or IL2 (500 units/ml) either alone or in combination for 48 h. For blocking peptide assay, cells were treated with SN52 or SN52M (50 µM/ml) for 30 min prior to the above treatment. Cells were washed and stained with surface markers and fixed/permeabilised according to the manufacturer’s instruction. Then cells were stained with PE-conjugated control antibody or antibody to NFκB2 or Ki67 for 15 min at room temperature. Cells were washed, fixed and analysed promptly using a BD Fortessa flow cytometer.