Dmem low sugar medium

Recombinant rat macrophage-colony stimulating factor (M-CSF), interferon-γ (IFN-γ), and interleukin-4 (IL-4) were purchased from PeproTech (Rocky Hill, NJ, http://www.peprotech.com). LPS from Escherichia coli 055:B5 was obtained from Sigma-Aldrich (St. Louis, MO, http://www.sigmaaldrich.com). Antibodies included FITC anti-rat CD11b/c and PE anti-rat CD80 (BioLegend, San Diego, CA, http://www.biolegend.com), Alexa Fluor® 647 anti-rat CD163 (Bio-Rad, US, http://www.bio-rad.com/), Annexin V-Alexa Fluor647/PI Apoptosis Assay Kit (FMSAV647-100, FcMAS, Nanjing, China, http://www.fcmacs.com/), inducible nitric oxide synthase (iNOS) (R&D, MN, https://www.bio-techne.com/), and Arginase 1 (Arg-1) (CST, https://www.cellsignal.com/).
Cell culture-related reagents included fetal bovine serum (FBS) and DMEM low sugar medium (glucose content 1 g/ml; Gibco, US, http://www.thermofisher.com/cn), as well as penicillin/streptomycin and PBS (Hyclone, US, http://www.thermofisher.com/cn).

To measure the neutralizing activity of sera or IgGs against various SARS-CoV-2 variant pseudotyped viruses, 1 × 10⁴ 293T-ACE2 cells (100 μL) were seeded in 96-well plates in DMEM low-sugar medium (Gibco). The next day, the sera from HR121-immunized animals were three-fold serially diluted in another 96-well plate in a volume of 60 μL. Then, 60 μL SARS-CoV-2 pseudovirus (M.O.I. = 0.1) was added to the diluted sera and incubated for 1 h at 37 °C. Thereafter, 100 μL of the mixture was incubated with 293T-ACE2 cells for 24 h at 37 °C, and the supernatant was removed from the cells after incubation. Renilla luciferase activity was determined using a Renilla luciferase assay kit (Promega, Madison, WI, USA). The NT_50s of the sera or IC₅₀s of the IgGs were calculated in GraphPad Prism 8.0.1 software as mentioned above.