56 Chapter 2 were adjusted to the lowest protein concentration value. Lysates were then processed as previously described71. High resolution respirometry Hepatic organoids were collected in Ad.DMEM/F12 at 4°C and kept on ice for 10 minutes to properly disrupt the Matrigel. After centrifugation at 200g for 5 minutes at 4°C, supernatant was removed and organoids were washed with MiR05 buffer followed by another centrifugation step. Organoids were then resuspended in 600uL of MiR05 buffer (110mM sucrose, 60mM potassium lactobionate, 20mM taurine, 20mM HEPES, 0.5mM EGTA, 10mM KH2PO4, 3mM MgCl2, 1mg/ml bovine serum albumin, pH 7.1). The rate of oxygen consumption of the organoids was measured using a two-channel high-resolution Oroboros Oxygraph-2 k (Oroboros, Innsbruck, Austria) at 37°C. Firstly, organoids were provided with 1mM ADP. Maximal ADP-stimulated respiration was achieved by addition of the oxidable substrates Palmitoyl Carnitine (25uM) plus Malate (2mM) to stimulate the fatty acid β-oxidation (State 3). Next, basal respiration (state 4) was determined using Oligomycin to block ATP synthase. Finally, 0.5mM FCCP was added in order to study the oxygen consumption during uncoupled respiration (State U). Oxygen consumption rates were normalized for protein content. Mitochondrial copy number Organoids were collected in basal AdvDMEM/F12 and lysed in lysis buffer (100 mM Tris-HCL pH8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl and 20 mg/ml proteinase K ) for 2 hours prior to the addition of RNase A. Isopropanol was used to precipitate the DNA, and the pellet was washed with 70% ethanol. After centrifugation at max. speed at 4°C, the pellet was resuspended in TE buffer and DNA yields were measured. qPCR reactions were performed using FastStart Universal SYBR Green Master (Rox) (Sigma Aldrich) in 384 well format in duplicate with 10 ng (for the intestinal samples) and 20ng (for the hepatic samples) total DNA per well in a QuantStudio 7 Flex. Mitochondrial DNA was measured using home-made primers (Supplementary Table 2) and normalized to the values of nuclear β-globulin as reference.
RkJQdWJsaXNoZXIy MTk4NDMw