Phenol red free rpmi

CD4⁺ and CD8⁺ T-cells were cultured at 0.5 × 10⁶ cells/500 μl of phenol red free RPMI (Sigma) + 2 mM GlutaMAX (ThermoFisher). T-cells were cultured with 2-deoxy-d-glucose (25 mM) or oligomycin (1 µM) at 37°C in 5% CO₂-in-air for 24 h. All chemicals were purchased from Sigma. To prevent impaired T-cell activation, after 3 h 5% fetal bovine serum (FBS, HyClone, ThermoFisher Scientific) was added. Cells were analyzed via flow cytometry for cell death (DRAQ7) and activation (CD69); the supernatant was removed and stored at −20°C for downstream cytokine analysis. IFNγ and IL-2 were analyzed using ELISA as per manufacturer’s instructions (DuoSets; R&D Systems).

LNCaP cells were cultured in 6‐well plates at a density of 4 × 10⁵ cells/well in hormone‐depleted medium, comprising phenol red‐free RPMI (Sigma) supplemented as above but with 5% charcoal‐stripped fetal bovine serum (Labtech International), for 72 h prior to treatment with 10 nM mibolerone (Mib) (PerkinElmer Life Sciences, Wellesley, MA), 1 μM bicalutamide (Bic) (Sigma, Chemical Co., St. Louis, MO) or 10 nM 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG) (Sigma). Cells were harvested for protein extraction and protein expression assessed by Western blotting using 10 μg of total lysate.

The number and percentage of dead cells based on plasma membrane integrity of the adherent cell population was quantified by analysis of microscopy images. Cells in 96-well flat bottom plates (30,000 cells/well) were stained with 2 µg/ml propidium iodide (Sigma-Aldrich) and 2 µg/ml Hoechst 33342 (H3570, Sigma-Aldrich) in 40 µl/well phenol red-free RPMI (Sigma-Aldrich) supplemented with 10% FBS and 2 mM L-Alanyl-L-Glutamine and incubated for 5 min at room temperature (RT). Cells were imaged using a Leica AF6000 LC fluorescence microscope (Leica Microsystems, Wetzlar, Germany) combined with a 10x dry objective. Total and dead cell numbers were quantified by respectively counting the nuclei and the number of propidium iodide-positive cells using ImageJ software (64 (link)). STSP (2.5 µM) was included as a positive control for cell death (Supplemental Figure 4).