Isotype matched iggs

Human SDSCs were expanded on either Plastic or dECM deposited by either hSDSCs (HECM) or pSDSCs (PECM) followed by the evaluation of surface marker expression for CD45 using anti-CD45 APC (BD Biosciences) and HLA-DR [major histocompatibility complex (MHC), class II, DR] using anti-human HLA-DR FITC (eBioscience, San Diego, CA), and isotype-matched IgGs (Beckman Coulter). Samples (n=5) of each 3×10⁵ expanded cells were incubated on ice in cold PBS containing 0.1% Chrom-Pure Human IgG whole molecule (Jackson ImmunoResearch Laboratories) and 1% NaN3 (Sigma-Aldrich) for 30 min. The cells were then incubated in the dark in the primary antibodies (both HLA-DR and CD45 simultaneously) for 30 min. The fluorescence data were analyzed by a BD LSRFortessa^™ (BD Biosciences), collected using BD FACSDiva 8.0 Software (BD Biosciences) at 10,000 events/sample. The results were analyzed using FCS Express 4 software (De Novo Software).

The following primary antibodies were used to detect expanded cell surface profiles: CD29 (abcam, Cambridge, MA), CD90 (BD Pharmingen, San Jose, CA), CD105 (BioLegend, San Diego, CA), the stage-specific embryonic antigen 4 (SSEA4) (BioLegend), and isotype-matched IgGs (Beckman Coulter, Fullerton, CA). The secondary antibody was goat anti-mouse IgG (H + L) R-phycoerythrin conjugated (ThermoFisher Scientific, Milford, MA). Samples (n=3) of each 2×10⁵ expanded cells were incubated on ice in cold PBS containing 0.1% ChromPure Human IgG whole molecule (Jackson ImmunoResearch Laboratories, West Grove, PA) and 1% NaN3 (Sigma-Aldrich) for 30 min. The cells were then sequentially incubated in the dark in the primary and secondary antibodies for 30 min. Fluorescence was analyzed by a FACS Calibur (BD Biosciences) using FCS Express software package (De Novo Software, Los Angeles, CA).