Hepatocytes were isolated from male Wistar rats fasted for 16 h by collagenase digestion as described previously13 . Isolated hepatocytes (>90% viable by the trypan blue exclusion test) were seeded on 24‐well plastic plates (Corning, New York, NY, USA) at a density of 5.0 × 105 cells per well. Cells were cultured in Krebs–Ringer buffer and incubated in 95% O2/5% CO2 at 37°C for 2 h in the presence of 0.24 mmol/L 3‐isobutyl‐1‐methylxanthine and gluconeogenic precursors (10 mmol/L lactate and 1 mmol/L pyruvate) with/without LPS (0.02−20.0 μg/mL). After the incubation, medium was centrifuged at 50 g for 3 min to separate supernatants and pellets.
Splenocytes were harvested from the spleens of rats by homogenization. Crude cells were left to sink to the bottom of tubes for 1 min, and the supernatant was then centrifuged at 500 g for 3 min. Ammonium chloride was added to the precipitate in order to lyse red blood cells. Then, 30 s later, cells were washed twice with Dulbecco's Modified Eagle's Medium (DMEM). Isolated splenocytes were seeded on 10‐cm plastic dishes at a density of 1.0 × 106 cells/mL, and cultured in DMEM supplemented with 100 μmol/L 2‐mercaptoethanol with/without LPS (0.01–1.0 μg/mL).
Splenocytes were harvested from the spleens of rats by homogenization. Crude cells were left to sink to the bottom of tubes for 1 min, and the supernatant was then centrifuged at 500 g for 3 min. Ammonium chloride was added to the precipitate in order to lyse red blood cells. Then, 30 s later, cells were washed twice with Dulbecco's Modified Eagle's Medium (DMEM). Isolated splenocytes were seeded on 10‐cm plastic dishes at a density of 1.0 × 106 cells/mL, and cultured in DMEM supplemented with 100 μmol/L 2‐mercaptoethanol with/without LPS (0.01–1.0 μg/mL).