Primary hepatocytes from 12–16-week-old Balb/c or C57BL/6 mice were isolated as described previously (Prado et al., 2015). Briefly, immediately after euthanizing the mouse, the liver was sequentially perfused via the portal vein with HEPES buffer for 10 min and 100 U/ml collagen prewarmed to 37°C. Afterwards, the inferior vena cava was incised to drain the blood, circulating cells and the perforate from liver. The inferior vena cava was periodically clamped for 10 s to ensure optimal distribution of the perfusion solutions. From the excised liver, the tissue capsule surrounding the liver was disrupted to release liver cells into wash medium (William medium E containing 2 mM L-glutamine) by gently shaking. The hepatocytes were separated from other liver cells by centrifugation at 50 g for 2 min and washed two more times in wash medium at same centrifugation settings. Viability of the isolated hepatocytes was assessed using Trypan Blue dye exclusion. 100,000 to 200,000 primary hepatocytes were seeded on cover slips and cultured in William medium E containing 2 mM L-glutamine, 10% FCS and antibiotics (100 U penicillin and 100 µg/ml streptomycin) at 37°C and 5% CO2. Before continuing with P. berghei infection on the next day, the growth medium was changed.

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