Mouse monoclonal igg2a

All the chemicals and reagents used were of the highest purity available. Phenylmethanesulfonylfluoride, β–mercaptoethanesulfonic acid sodium salt, β–mercaptoethanol, Ethanolamine, Sepharose-4B, Freund's complete and incomplete adjuvant, Bicinchoninic acid (BCA) protein estimation kit, Tween-20 and FITC conjugated mouse anti-rabbit antibody were purchased from Sigma-Aldrich Chemicals (St Louis, MO) and used as received. Horseradish peroxidase-conjugated anti-rabbit IgG were purchased from Bangalore Genei (India) Pvt. Ltd. (Bangalore, India). Mouse monoclonal anti-Listeria LZH1 IgG1 and mouse monoclonal IgG2a specific for the human erythrocyte membrane protein (Protein4.2 (2G-12)) were procured from Santa Cruz Biotechnology, Inc., California and Abnova Corporation respectively.

WJMSCs were analyzed for the expression of surface antigens and DNA content using flow cytometer (BD FACS Calibur; Becton Dickinson, NJ) in triplicates from three independent experiments. For phenotyping of surface antigens, WJMSCs were harvested using 0.25% Trypsin‐EDTA and fixed in 3.7% formaldehyde solution. The cells were then washed twice with DPBS and labelled (1 × 10⁵ cells per marker) with fluorescein isothiocyanate‐conjugated CD34 (BD Pharmingen, CA, FITC Mouse Anti‐Human CD34), CD45 (Santa Cruz Biotechnology, FITC Mouse Anti‐Human CD45), CD90 (BD Pharmingen, FITC Mouse Anti‐Human CD90) and unconjugated CD73 (Santa Cruz Biotechnologies, Mouse monoclonal), and CD105 (Santa Cruz Biotechnologies, Mouse monoclonal IgG_2a) for 30 min. Unconjugated primary antibodies were treated with secondary FITC‐conjugated goat anti‐mouse IgG (BD Pharmingen) for 30 min in the dark. For isotype matched negative control Mouse IgG1 (BD Pharmingen) was used. A total of 10,000 labeled cells per sample were acquired and results were analyzed using cell Quest Pro software (Becton Dickinson). For evaluating DNA content, a total of 1 × 10⁶ cells/ml were fixed in 70% ethanol at 4°C for 4 h. After washing cells twice with DPBS, they were stained with 10 µg/ml PI solution for 15 min. DNA content of each cell was measured and categorized as G₀/G₁, S, or G₂/M phase of the cell cycle.