Culturing and Differentiating Cell Lines for SARS-CoV-2 Research

Vero CCL-81 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin-streptomycin, 4.5 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, and 1.5 g/L NaHCO₃. Calu-3 cells were cultured in DMEM medium supplemented with 20% FBS, 1% non-essential amino acids, 4.5 g/L glucose, 2 mM L -glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin-streptomycin and 1.5 g/L NaHCO₃. THP-1 cells were cultured in RPMI-1640 supplemented with 10% FBS, and 1% penicillin-streptomycin at 37 °C. All cells were kept in a humidified 5% CO₂ atmosphere. THP-1 monocyte cells were differentiated into macrophage-like cells as described by Gatto et al. [77 (link)] with few modifications. THP-1 monocytes were induced to differentiate into macrophages by the addition of phorbol-12-myristate 13-acetate (PMA, 50 ng/mL) (ab120297, Abcam, Cambridge, UK) for 48 h (h). After this time, the PMA-containing medium was replaced with fresh medium without PMA for 24 h prior to SARS-CoV-2 infection. Cell differentiation was verified by evaluating cell adhesion and spreading under an optical microscope.