Alexa fluor 488 f ab 2 fragment of goat anti mouse igg h l antibody

MARC-145 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing low glucose and low bicarbonate, while human embryonic kidney (HEK) 293T cells (ATCC^® CRL-3216^TM) were maintained in DMEM high-glucose (Life Technologies, Grand Island, NY, USA). 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) (herein designated as cDMEM). All cell lines were cultured at 37 °C in 5% CO₂ in an incubator. 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). Goat anti-mouse IgG (H+L)-HRP antibody and DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA).

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).