188 Chapter 6 Oxygen consumption rate was measured in the organoids using a two-channel high-resolution Oroboros Oxygraph-2 k (Oroboros). Organoids were first permeabilized by addition of digitonin (0.02 mg/ml). The maximal coupled respiration was measured in the presence of 1mM ADP, 25 μM octanoylcarnitine and 2mM malate (state 3). Basal respiration was determined by the subsequent addition of 0.002 mg/ml of oligomycin to block ATP synthase (state 4). Finally, uncoupled respiration (state U) was measured after subsequent administration of 1.5 μM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP). Oxygen consumption rates were normalized to protein concentration. Acylcarnitines measurements EHO organoids were incubated with 0.5mM BSA-conjugated palmitate (SigmaAldrich, P9767) and 2 mM l-carnitine (Sigma Aldrich) for 24 h prior to collection in PBS. Acylcarnitines were measured both in supernatant and intracellularly following the published protocol55. Total CoA measurements using HILIC-MS/MS analysis Mat-EHO organoids were incubated in glucose-free maturation medium supplemented with 0.5mM BSA-conjugated palmitate and 2 mM l-carnitine for 24 h. Organoids were collected in ice-cold medium and washed 2X in ice-cold PBS. Samples were prepared as described elsewhere56. Briefly, the pellet was reconstituted in 600 μL MilliQ H2O and the lysate was sonicated using a Sonics Vibra cell VCX130 (25 seconds, 50% amplitude, 2 times). Lysates were centrifuged at 14000 rpm for 15 minutes at 4°C. In a new tube, 80 μL Tris (2-carboxyethyl)phosphine hydrochloride (10 mM) was added to 400 μL supernatant and, incubated for 15 minutes at room temperature. Next, samples were spun down (14000 rpm, 15 minutes, 4°C). In a new tube, 40 μL ammonia solution was added to 400 μL supernatant (1.25% v/v) and, incubated shaking at 500 rpm at 60°C for 60 minutes. Lastly, samples were dried using a SpeedVac (Eppendorf) and reconstituted in 100 μL ice-cold 80% methanol. Coenzyme A was extracted by a two-step protocol using chloroform/methanol/ water based on the Bligh and Dyer approach57. The detailed sample preparation and HILIC-MS/MS protocol are described in detailed in the literature58.
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