Alexa 488 conjugated goat anti rabbit igg h l

Baby hamster kidney (BHK-21) cell line purchased from American Type Culture Collection (ATCC; Manassas, VA, USA) was cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Sigma, St Louis, MO, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL). The GI JEV strain of YZ-1 (GenBank accession number: MZ540901) and GIII JEV strain of HSY-1 (GenBank accession number: MZ540902) were isolated and stored in our laboratory, and all viruses were propagated and titrated in BHK-21 cells. A polyclonal rabbit antibody against JEV NS3 protein was purchased from Genetex (St. Anthony, TX, USA). Alexa 488-conjugated goat anti-rabbit IgG H&L was purchased from Jackson ImmunoResearch Inc. (West Grove, PA, USA). The low-copy plasmid pOK-12 was purchased from BioVector NTCC Inc. (Beijing, China).

The mBMSC culture and differentiation medium was purchased from Cyagen (China). The chondrocyte activator IL-1β was purchased from Sigma-Aldrich (USA). The primary antibodies used in this study included mouse anti-CD9, CD63, CD81, and SOX9 (Santa Cruz Biotechnology, USA); rabbit anti-COL II (Santa Cruze Biotechnology); rabbit anti-PDGFBB, VEGF, SDF-1, COL X, anti-cleaved caspase-3, Bcl-2, Bax, and GAPDH (Abcam, USA); rabbit anti-TGFβ1, Smad2/3, p-Smad2/3, EKR1/2, p-EKR1/2, p38, and p-p38 (Cell Signaling Technology, USA); rabbit anti- COL X and Ki67 (Novus, USA); rabbit anti-Aggrecan, and MMP13 (Proteintech; China).The secondary antibodies used in this study included Alexa-488 conjugated-goat anti-rabbit IgG (H + L) (Jackson ImmunoResearch, USA); horseradishperoxidase–conjugated-goat anti-rabbit IgG (H + L) and horseradish peroxidase–conjugated-goat anti-Mouse IgG (H + L) (Invitrogen, USA); Nuclei was stained with DAPI dihydrochloride (Thermo Fisher Scientific, USA). Flow cytometry anlysis was performed to identify the characterization of mBMSCs stained with FITC-conjugated or PE-conjugated anti-mouse CD44, CD45, CD90, and CD105 (BD, USA).