187 6 iPSC-derived liver organoids as a tool to study Medium Chain Acyl-CoA Dehydrogenase deficienc All results were normalized to protein content. Immunoblotting The protocol followed for immunoblotting was slightly modified from a previously published paper54. Organoids were collected using ice-cold Ad. DMEM/F12 and kept on ice for 10 minutes to degrade the BME. Organoids were then centrifuged at 290g for 5 minutes at 4°C and washed with cold PBS prior to another centrifugation step. After the second centrifugation, the organoids were reconstituted in radio immunoprecipitation assay (RIPA) buffer containing 1% IGEPAL CA-630, 0.1 % SDS, and 0.5 % sodium deoxycholate in PBS. RIPA buffer was supplemented with Phosphatase Inhibitor Cocktail 2 (Cat. No. P5726) and Cocktail 3 (Cat. No. P0044) and Complete Protease Inhibitor Cocktail (Cat. No. 1186145001) (All Sigma Aldrich). Sonics Vibra cell VCX130 (Sonics & Materials Inc.) was used to sonicate organoid lysates using 4 pulses of 10 second on, 30 seconds off at an amplitude of 30%. Lysates were then centrifuged at 12000 rcf for 10 minutes at 4°C to ensure the precipitation of cell debris. Protein content was determined using Pierce BCA Protein Assay Kit (ThermoScientific) and all samples were adjusted to the lowest concentration value. Lysates were adjusted with Laemmli loading buffer (5X: 60 mM Tris-Cl pH 6.8, 10% glycerol, 1% SDS, 0.05% Bromophenol Blue, 1% beta-mercaptoethanol). Protein separation was done in SDS-PAGE 10-14% using a Mini PROTEAN Tetra Vertical Electrophoresis Cell system (Bio-Rad, 1658029FC). For western blot, proteins were transferred to a polyvinylidene difluoride membrane (Immobilon®-P, Millipore). Fat isolation and triglyceride quantification Organoids were collected in 1x TBS (137 mM NaCl, 2.7 mM KCl, 66 mM Tris, pH 7.4) in MiliQ water. Fat was extracted in chloroform: methanol in a ratio 2:1. The levels of hepatic triglycerides were quantitatively determined using the DiaSys Triglyceride FS kit (Holzheim). Results were normalized to protein content. High resolution respirometry Organoids were collected at day 8-10 using ice-cold Ad. DMEM/12 and kept on ice for 10 minutes. Organoids were then centrifuged at 290 g at 4°C for 5 minutes and washed with 2mL of MiR05 buffer, followed by another spin and finally reconstituted in 600uL of MiR05 buffer, containing 110 mM sucrose, 60 mM potassium lactobionate, 20 mM taurine, 20 mM HEPES, 0.5 mM EGTA, 10 mM KH2PO4, 3 mM MgCl2, and 1 mg/ml bovine serum albumin, at pH 7.1.
RkJQdWJsaXNoZXIy MTk4NDMw