109 4 Docosahexaenoic acid prevents peroxisomal and mitochondrial protein loss in a murine hepatic organoid model after 24 h and 48 h and filtered through a 0.45 μm SFCA filter (Corning, Cat N: 431220). Finally, 0.5 μL of polybrene (4mg/mL stock concentration) (SIGMA, Cat N: H9268-5G) per mL of media was added to enhance retroviral transduction. For transduction of target cells, 1 mL or 2 mL (depending on experiment design) of retroviral supernatant was used. Then, target cells were incubated for at least 24 hours at 37oC and 5% CO2. Establishment of LC3B-GFP-RFP-LC3ΔG liver organoids Organoids were collected in Advanced DMEM/F12 and kept on ice for 10 minutes to disrupt the Matrigel. Organoids were then centrifuged at 290G for 5 minutes and reconstituted in 1mL of pre-warmed Trypsin for 5 minutes. Then, mechanical disruption of the organoids was used to dissociate them into single cells. 10 mL of advanced DMEM/F12 were added and the organoids were centrifuged again to remove the trypsin. Cells were counted and approximately 4*105 cells were placed in 500 µL of expansion medium supplemented with 10 µM Y 27632 dihydrochloride. Equal volume of viral medium, produced as described in the previous section, was added and placed into a 24 well plate well (pre-coated with Matrigel). The cells were kept in the incubator at 37oC for 48 h. Cells were then trypsinized and collected. After removal of the trypsin, the cells were reconstituted in advanced DMEM/F12 and Matrigel (1:3) and plated in domes. Expansion medium was supplemented with 10 µM Y 27632 dihydrochloride. After 24 h, the medium was changed to selection medium (EM containing Puromycin 1:500) FACS sorting of stable mouse liver organoids expressing the autophagic flux probe In order to isolate single clonal progenitors of liver organoids that properly express the autophagic flux probe GFP-LC3-RFP-LC3ΔG (double positive cells) and exclude GFP-LC3ΔG cells caused by homologous recombination during the transfection and transduction procedures reported by Kaizuka et al., 201636 we sorted the polyclonal stable liver organoids and performed an enrichment of GFP-RFP double positive cells. In brief, polyclonal liver organoids were harvested and individualized as previously mentioned. Cells were kept on ice in FACS buffer containing 1X HBSS (Gibco), 2% FCS (Gibco), 2% BSA, 1% Penicillin-Streptomycin (P/S). Cell sorting was performed using the MoFlo Astrios EQ Cell Sorter (Beckman Coulter, Life sciences) with a 100 μm nozzle and the 488 nm and 561 nm lasers. Sort rate was set up to 25.000 events/ sec and approximately 1.000.000 double positive events were recorded and
RkJQdWJsaXNoZXIy MTk4NDMw