Goat anti human cd163 polyclonal antibody

MARC-145 and PAM cells were seeded in a 48-well plate at the cell density of 5 × 10⁴ per well. One day later, the cells were first incubated either with 10 μg of goat anti-human CD163 polyclonal antibody (R&D System, Minneapolis, MN, USA) or normal goat IgG diluted in 100μL of culture medium for 1 h at 37 °C. Subsequently, the treated cells were inoculated with PRRSV strains at an MOI of 2.5 TCID50 per cell. After 1 h adsorption at 37 °C, the inoculum was removed, and cells were washed thrice with the culture medium and supplemented with 500 μL of cDMEM or cRPMI-1640 medium. At 12 hpi, the culture supernatants were collected to determine the viral genome copy by RT-PCR and the cells were analyzed by IFA and flowcytometry to quantify the percentage of PRRSV-infected cells.

MARC-145, a monkey kidney cell line, was cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing low glucose and low bicarbonate. PK15-pCD163 (kindly provided by Dr. X.J Meng at Virginia Polytechnic Institute and State University) and HEK-293T cells were maintained in DMEM high glucose. PAMs used for the ex vivo infections were isolated from the lung lavage of pigs between 4- or 8-weeks-old. All media were supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO, USA), 100 units/mL of penicillin, and 100 μg/mL of streptomycin (Sigma, St. Louis, MO, USA). All cell types used in this study were cultured at 37 °C, with 5% CO₂. The mouse monoclonal antibody SDOW17 specific to PRRSV N protein was purchased from the National Veterinary Services Laboratories (Ames, IA, USA). Alexa Fluor-488 F(ab’)2 fragment of goat anti-mouse IgG (H+L) antibody was purchased from Invitrogen (Eugene, OR, USA). Fluorescein isothiocyanate (FITC) conjugated mouse anti-PRRSV N protein antibody (clone SR30) was purchased from Rural Tech lnc., (Brookings, SD, USA). Goat anti-human CD163 polyclonal antibody was purchased from R&D system (clone AF1407, Minneapolis, MN, USA). Goat anti-mouse IgG (H+L)-HRP antibody and DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) were purchased from ThermoFisher Scientific (Carlsbad, CA, USA).