Cd105 apc

To determine the nature of cultured cells, isolated cells were immunostained with the antibody against STRO-1 (1 : 200, Novus Biologicals, USA) and cytokeratin (1 : 100, Bioworld, USA). Phosphate buffered saline (PBS) was simultaneously used as a control. Flow cytometric analysis of specific surface antigens was also used to characterize the cultured cells. Cells were harvested and incubated with various combinations of the following fluorochrome-conjugated rabbit anti-human antibodies: CD34-FITC, CD45-PerCP, CD90-PE, CD105-APC, CD146-APC, and CD73-PE (all from Miltenyi, Germany) for 20 min at room temperature in the dark. The corresponding mouse IgG isotype control antibodies conjugated to FITC, PE, APC, or PerCP were employed as negative controls in each experiment. Stained cells were washed twice with 0.01 mol/L PBS and analyzed using BD FACSCalibur (BD Biosciences, USA).

Pre-conjugated antibodies CD49c-APC and CD166-PE were purchased from BioLegend, San Diego, CA, USA. SSEA4-PE, CD29-APC, CD54-PE, CD90-FITC, CD105-APC, CD106-APC were purchased from Miltenyi Biotec Inc., San Diego, CA, USA. Isotype IgG control antibodies were also purchased from Miltenyi Biotec Inc. Cells to be stained were washed 2 times with 5.0 mL of sterile HBSS and detached using 2.0 mL of TrypLE Express (Life Technologies, Grand Island, NY, USA). Cells were washed with DMEM supplemented with 10% FBS and spun down using a centrifuge set for 300xg. Cells were washed once again with 5.0 mL sterile 1x PBS and spun down at 300xg. Viable cell number was quantified using a hemacytometer and 0.4% Trypan blue solution (Life Technologies, Grand Island, NY, USA). For each sample to be stained, 1.0 × 10⁶ viable cells were resuspended in 100 µL of Flow buffer (1x PBS, pH 7.2, 0.5% bovine serum albumin and 2 mM EDTA). Pre-conjugated antibody (10 µL) was added to the resuspension, mixed and incubated for 10 min in the dark at 4 °C. Cells were washed 3 times with 1.0 mL of 1x PBS and resuspended in 500 mL of Flow buffer before single channel FACS analysis using an Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA). Control experiments for non-specific staining using mouse IgG were performed alongside all experiments.

STRO-1 is a protein-tagged gene of MSCs, which is the first isolated monoclonal antibody to identify MSCs. Cultured cells (3 days) were subjected to immunofluorescence staining with an antibody of STRO-1 (1:200, Novus Biologicals, Littleton, CO, USA), followed by determination of positive expression of STRO-1. Meanwhile, cells were incubated with CD34-FITC, CD45-PerCP, CD90-PE, CD105-APC, and CD73-PE (Miltenyi, Bergisch Gladbach, Germany) and subjected to FCM analysis (BD Biosciences, CA, USA).