110 Chapter 4 harvested in a FACS tube containing 2 mL of expansion medium supplemented with 10µM Y 27632 dihydrochloride and 2μg/mL puromycin selection treatment followed by a centrifugation step at 290G for 5 minutes; then the pellet was reconstituted in 1 mL of pre-warmed expansion medium supplemented with puromycin (2 μg/mL). Cells were counted and viability was determined by trypan blue staining (higher than 90%). Approximately 5000 sorted cells were reconstituted in advanced DMEM/F12 and Matrigel (1:3 ratio) and plated in domes in 24 well plates. The organoids were maintained in 500 μL expansion medium supplemented with 10 µM Y 27632 dihydrochloride and 2 μg/mL puromycin selection treatment and kept in the incubator at 37oC and 5% CO2. Enough liver progenitors were seeded and expanded for either performing experiments or storage in liquid nitrogen. Two days after sorting the formation of new liver organoids was confirmed as well as the expression of GFP and RFP were determined by fluorescence microscopy. Generation of stable HuH7 cells expressing the autophagic flux probe Wild-type HuH7 cells were seeded in control medium containing DMEM supplemented with 1% P/S and 10% FCS in a 6 well plate a day before transduction and kept in the incubator at 37oC and 5% CO 2.Cells were transduced with 2 mL of retroviral supernatant and incubated for 48 h. Next day and 48h after transduction cells were checked for positive GFP and RFP expression by fluorescence microscopy. After 48 h post-transduction, cells were harvested and expanded for two consecutive passages. The transduced-HuH7 cells were maintained in DMEM-GlutaMAX (Gibco, NL) media supplemented with 10% FCS, 1% P/S and 2 μg/mL puromycin. The cells generated in this step were considered polyclonal stable HuH7 cells expressing the autophagic flux probe GFP-LC3-RFP-LC3ΔG, in short cells were called Poly-HuH7-3ΔG. In order to produce single monoclonal HuH7 cells that properly express the autophagic flux probe GFP-LC3-RFP-LC3ΔG (double-positive cells) and exclude homologous recombination during the transfection and transduction procedures reported by Kaizuka et al., 201636 we sorted the polyclonal cells. In short, polyclonal HuH7 cells were harvested and kept on ice in FACS buffer containing 1X HBSS, 2% FCS, 2% BSA, 1% P/S. Cell sorting was performed using the MoFlo Astrios EQ Cell Sorter (Beckman Coulter, Life sciences) with a 100 μm nozzle equipped with the 488 nm and 561 nm lasers. Sort rate was set up to 25.000 events/ sec and individual sort mode was used. Single double-positive (GFP/RFP) cells were dripped in 96-well plates previously filled with 200 μL of standard cultured media supplemented with 2 μg/mL puromycin. A total of 3 96-well
RkJQdWJsaXNoZXIy MTk4NDMw