Pe conjugated anti cd105

Phenotype of cultured cells was analyzed using directly labeled monoclonal mouse antibodies: PE-conjugated anti-CD133 and FITC-conjugated anti-CD271 (Miltenyi Biotec, Germany); PE-conjugated anti-CD90 (BD Biosciences, USA); PE-conjugated anti-CD105, PE-conjugated anti-HLA-DR (BD Pharmingen) and anti-CD146 (R&D Systems, USA). All antibodies were used at the concentration of 1 to 5 µl of antibody for 1 × 10⁵ cells in 50 µl of PBS supplemented with 1% FBS, as suggested by the manufacturer, and incubated for 30 min at 4 °C. After incubation cells were carefully washed. IgG isotype matched, PE or FITC labeled immunoglobulins were used as controls. Cells were analyzed by flow cytometry using FACSCalibur (Becton Dickinson, CA). Data were presented using WinMDI 2.7 software.

The cultured MSCs were washed in PBS, trypsinized, washed in PBS-5% FBS and incubated with FITC-conjugated anti-CD3(BD Biosciences,553733), CD29(BD Biosciences,55500), CD117(BD Biosciences,561680) or PE-conjugated anti-CD105(BD Biosciences,562759), CD44(BD Biosciences,561860) and Sca-1(BD Biosciences,553108) antibodies (10 μg/mL in PBS-0.5% bovine serum albumin) for 1 h, then washed 3 times with PBS. All incubations were performed at 4°C. MSCs, MSCs with FITC and PE dye without antibodies were used as controls. Lastly, all the cells were analyzed on a FACS can instrument (Attune® Acoustic Focusing Cytometer, Applied Biosystems)[28 (link)]