Hepes

Rates of oxygen consumption (OCR) and extracellular acidification (ECAR) were assessed using a Mitochondrial Stress Test on an Agilent XFe96 Seahorse analyzer. An XFe96 Spheroid Microplate (Agilent) was coated with 40 µL of 1% Fibronectin in dPBS for 1 h at 37 °C. Spheroids were seeded on the coated plate at densities of 10, 20, and 30 spheroids and incubated at 37 °C overnight in CDM3 medium. The following day, media was exchanged for Seahorse XF RPMI media containing 1 mM HEPES (Agilent Technologies, Santa Clara, CA, USA) and supplemented with 2 mM glutamine and 11 mM glucose. Indices of mitochondria function were assessed by sequential addition of 1 µg/mL Oligomycin, 1 µM FCCP, and 1 µM Rotenone/Antimycin A to quantify rates of basal/maximal OCR and spare respiratory capacity. Assay results were normalized to total protein content quantified using a Pierce™ Coomassie (Bradford) Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) following lysis with M-PER™ Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockford, IL, USA).

Proton efflux rate (PER) analysis was performed on a Seahorse XFe96 Analyzer (Agilent Technologies, Santa Clara, CA, USA) using Agilent Seahorse XF Glycolytic Rate Assay kit, following the manufacturer's instructions. Before the assay, FL^MycER cells were treated with OHT and IACS‐010759 for 72 and 24 h as indicated. The cells were then counted and attached to 96‐well Seahorse cell culture microplates, precoated with Corning™ Cell‐Tak (Life Sciences) according to the manufacturer's instructions, at a density of 80,000 cells per well, in XF RPMI Medium pH 7.4 with 1 mM HEPES (Agilent Technologies) supplemented with 2.75 mM glucose, 1 mM sodium pyruvate, and 2 mM L‐glutamine. The plate was incubated at 37°C for 1 h in a non‐CO₂ incubator. After ECAR baseline measurements, 0.5 μM rotenone plus 0.5 μM antimycin A and 50 mM 2‐deoxyglucose (2‐DG) were added sequentially to each well. The results were analyzed using the Seahorse Wave Desktop Software Version 2.6 (Agilent Technologies) and normalized by cell number using CyQUANT Cell Proliferation Assay (Thermo Fisher Scientific). Data were exported into the XF Report Generator for calculation of the parameters from the Glycolytic Rate Assay. Results are mean ± SD of minimum eight technical replicates and representative of two independent experiments.

Mitochondrial function (oxidative phosphorylation) and glycolytic rate of naïve bone marrow monocytes were assessed using a modified variant of the XFp Cell Mito Stress Kit and a Seahorse XFp Analyzer (both Agilent Technologies, Waldbronn, Germany). In brief, monocytes were washed with Seahorse XF Base Medium (Agilent Technologies, Waldbronn, Germany) supplemented with 10 mM glucose (Sigma Aldrich, Taufkirchen, Germany), 5 mM HEPES (Agilent Technologies, Waldbronn, Germany), 2 mM glutamine (Life Technologies, Darmstadt, Germany) and 1 mM pyruvate (Life Technologies, Darmstadt, Germany), and seeded in Cell-Tak (Corning, Kaiserslautern, Germany) coated 8-well Seahorse plates (1.5 × 10⁵ cells per well). All experiments were performed with two technical replicates. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured consecutively three times under basal conditions and after sequential injection of Oligomycin A (1 μM), FCCP (2 μM), Rotenone/Antimycin A (0.5 μM; all three reagents included in XFp Cell Mito Stress Kit, Agilent Technologies, Waldbronn, Germany) and finally 2-Deoxyglucose (50 mM) (Sigma Aldrich, Taufkirchen, Germany). Evaluation and calculation of mitochondrial and glycolytic indices was done using Wave software (Agilent Technologies, Waldbronn, Germany).