Paraformaldehyde pfa pbs

HEK293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s media (DMEM; Gibco-Life Technologies, Grand Island, NY, USA) with 4.5 g/L glucose (25 mM), 10% fetal bovine serum (FBS; Gibco-Life Technologies), 1% penicillin–streptomycin (Pen-Strep; Gibco-Life Technologies) and 5 μg/mL plasmocin (InvivoGen, San Diego, CA, USA) at 37 °C with 5% CO₂. For osmotic stress, HEK293T cells were exposed to 400 mM of sorbitol (Sigma, St. Louis, MO, USA) for 4 h. For recovery assays, cells were washed with PBS once and maintained in DMEM for 4 h. To induce proteasome inhibition, cells were incubated with 10 µM of MG132 for 4 h. For fluorescence in situ hybridization (FISH) and immunofluorescence, cells were seeded on coverslips coated with attachment factor protein (Gibco-Life Technologies). Cells on coverslips were fixed with 4% paraformaldehyde (PFA)-PBS (Electron Microscopy Sciences, Hatfield, PA, USA, 32%) solution for 10 min and washed with PBS.

NC-iPSC and C-iPSC were plated in three of four wells of a four-well chamber slide and differentiated to HLC (see above). Three wells were treated overnight in LPDS medium supplemented with 5 µM rosuvastatin. The following day, two wells in rosuvastatin were treated with 10 µg/ml DiI-LDL (Thermo Fisher Scientific) for 6 h or 24 h while the remaining well did not receive any DiI-LDL. Cells were fixed with 2% paraformaldehyde (PFA)/PBS (10 min, 24°C; Electron Microscopy Sciences, Hatfield, PA, USA) and mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Slides were imaged using an Olympus IX81 fluorescence or FV1000 confocal microscope (Center Valley, PA, USA).