Murine Macrophage Response to Coronavirus

Primary cultures of bone marrow-derived macrophages (BMDMs) were established from 7–10-week-old C57BL/6J mice, as previously described [29 (link)]. On the 6th culture day, BMDMs were resuspended in L929-conditioned medium (5% L929 supernatant, 10% fetal bovine serum [Gibco, Carlsbad, CA, USA] and 2 mM L-glutamine [Gibco, USA] in RPMI 1640 [CultLab, São Paulo, Brazil]) and seeded at 2.5 × 10⁵ cell density onto 24-well plates. On the next day, BMDMs were washed with PBS and inoculated with 150 µL serum-free RPMI 1640 loaded with either MHV-3 or MHV-A59 at 0.1 MOI. After 1 h of viral adsorption, cells were washed in PBS and cultured for 24 h with 0.5 mL of fresh RPMI 1640 containing calcitriol (Cayman Chemical Co., Ann Arbor, MI, USA) at different concentrations (0.1, 1.0, 2.5, or 5.0 µM) or 0.01% ethanol at 100% (vehicle, corresponding to the 5.0 µM dose of calcitriol). Virus yields were quantified in the supernatant via plaque assay. Cell damage was assessed indirectly by quantifying the abundance/activity of lactate dehydrogenase (LDH) in the supernatant via spectrophotometric measurements (catalog no. K014, Bioclin, Belo Horizonte, Brazil).