55 2 Organoids as a model to study intestinal and liver dysfunction in severe malnutrition BODIPY Staining Hepatic organoids were fixed in 4% PFA for 1 hour at 37°C. Permeabilization and staining were done simultaneously in CLMS buffer (PBS, 3% BSA, 10mM glycine) containing 0.1% saponine, 15μM BODIPY (Thermo Fisher Scientific) and 5µM Hoechst (Thermo Fisher Scientific) for 1 hour at room temperature. The organoids were washed with PBS and images were acquired with an AxioObserver Z1 compound microscope, 40X objective and AxioCam Mrm3 CCD camera. Permeability measurement (FITC) Permeability of intestinal organoids was assessed using fluorescence markers. Organoids were incubated with FITC-dextran 4kDa or 10kDa (TdB consultancy, Uppsala, Sweden) at a final concentration of 1.25µM in their respective Ad.DMEM/F12 for 30 minutes at room temperature. Organoids were washed 5-10 times with PBS to remove non-specific binding. The degree of accumulation in the lumen of 15 organoids chosen at random served as a measure for permeability. Organoids were imaged with an AxioObserver Z1 compound microscope, 2.5x or 10x objective and AxioCam MRm3 CCD camera. ZEN 3.1 blue was used to measure the fluorescence in the organoid lumen. To correct for differences in background fluorescence, the intensity was measured in three different areas around the organoid. The mean intensity measured in three squares around the organoid was subtracted from the luminal fluorescence. Immunoblotting For Western blotting, the protocol was based on a previously published paper71. Organoids were collected using the respective cold Advanced DMEM/F12 (with or w/o amino acids) and kept on ice for 10 minutes to ensure Matrigel dissolvement. Organoids were centrifuged at 200-290G for 5 minutes followed by one wash with cold PBS. Organoids were again centrifuged and resuspended in 200uL of radio immunoprecipitation assay buffer (1%IGEPAL CA-630, 0.1% SDS, and 0.5% sodium deoxycholate in PBS) supplemented with Complete Protease Inhibitor Cocktail (Cat. No. 1186145001; Sigma-Aldrich), Phosphatase Inhibitor Cocktail 2 (Cat. No. P5726; Sigma Aldrich) and Cocktail 3 (Cat. No. P0044; Sigma Aldrich). Organoid lysates were sonicated using Sonics Vibra cell VCX130 (Sonics & Materials inc, Newtown, USA) using a 50% amplitude four times for 10 seconds. Protein concentration was measured using Pierce BCA Protein Assay Kit (ThermoScientific, Rockford, IL, USA). All samples
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