Fitc conjugated anti cd34

Cultured human BM-MSC were harvested with TrypLE Select, and 1 × 10⁶ cells were incubated with primary antibodies for 30 min at 4 °C. The primary antibodies used in this study were FITC-conjugated anti-CD105, PE-conjugated anti-CD73, FITC-conjugated anti-CD90, FITC-conjugated anti-CD45, FITC-conjugated anti-CD34, PE-conjugated anti-CD11b, and PE-conjugated anti-CD14, FITC-conjugated anti-CD19 (BD Bioscience). Cells were washed with blocking reagent and analyzed in a FACS Calibur Flow Cytometer (Beckton Dickinson).

The whole blood was stained with APC-conjugated anti-Ter119 and PE-conjugated anti-CD61 for erythrocyte and platelet analysis. White blood cells after RBC lyses were stained with PE-conjugated anti-CD8, PE-Cy7–conjugated anti-B220, APC-conjugated anti–Mac-1, APC-Cy7–conjugated anti-CD4, and Pacific blue (PB)–conjugated anti-Gr1 (BioLegend). Single cell suspensions from BM and spleen were stained with lineage-marker cocktail followed by FITC-conjugated anti-CD41, PE-conjugated anti-CD150, PE-Cy7–conjugated anti-CD48, APC-conjugated anti–c-Kit, BV421-conjugated anti–Sca-1, and streptavidin-APC-Cy7. For progenitor analysis, cells were stained with lineage-marker cocktail followed by FITC-conjugated anti-CD34 (BD), PE-conjugated anti-CD16/32, APC-conjugated anti–c-Kit, BV421-conjugated anti–Sca-1, and streptavidin-APC-Cy7 antibodies (BioLegend). For megakaryocytes and erythrocyte precursor analysis, cells were stained with FITC-conjugated anti-CD42c (Emfret), Dylight-647–conjugated anti-αIIbβ3 (CD41/61; Emfret), PE-conjugated anti-CD71, and APC-conjugated anti-Ter119. Analyses were performed on an LSRFortessa (BD) or CyAn ADP Analyzer (Beckman Coulter), and results were analyzed with FlowJo software.

To fulfill the criteria of the International Society for Cellular Therapy and to exclude contamination of mesenchymal stem cell (MSC) cultures by hematopoietic cells, MSCs were analyzed by flow cytometry. Five antibodies—fluorescein isothiocyanate (FITC)-conjugated anti-CD34, PerCP-conjugated anti-CD45, allophycocyanin(APC)-conjugated anti-CD105, PC7-conjugated anti-CD90, and PE-conjugated anti-CD73 antibodies (BD Biosciences)—were used to identify the MSCs.21 22 (link)

Cells were trypsinized and resuspended in washing buffer consisting of PBS with 2.5% FBS, and stained with primary antibodies for 30 min at 4 °C. Cells were then stained with streptavidin-PE/Cy5 (1 : 200; BD Pharmingen, Franklin Lakes, NJ, USA) for 30 min at 4 °C in the dark. Primary antibodies used for cell staining were PE-conjugated anti-CD56 (1 : 20; Miltenyi Biotec), biotinylated anti-PDGFRα (2.5 μg/ml; R&D; cat. no. BAF322), FITC-conjugated anti-CD34 (1 : 10; BD Pharmingen), FITC-conjugated anti-CD45 (1 : 10; BD Pharmingen), FITC-conjugated anti-CD90 (1 : 200; BD Pharmingen), FITC-conjugated anti-CD105 (1 : 10; BioLegend, San Diego, CA, USA), and FITC-conjugated anti-CD166 (1 : 20; MBL, Aichi, Japan). Stained cells were analyzed by FACSCalibur or FACSVantage SE (BD Biosciences). Cell sorting was performed on a FACSVantage SE.