Apc conjugated mouse anti human cd49d clone 9f10

Cells were harvested by repeated pipetting after three days, washed in PBS (1x), and stained with monoclonal antibodies for FACS analysis. MSC from the different culture conditions were trypsinized, washed twice with PBS 1x, and stained with different monoclonal antibodies for flow cytometry analysis: PE-conjugated mouse anti-human CD49e (clone IIA1, BD Pharmingen, San Jose, CA, USA), APC-conjugated mouse anti-human CD49d (clone 9F10, BD Pharmingen, San Jose, CA, USA), APC-conjugated mouse anti-human CD54 (clone REA266, Miltenyi Biotec, Auburn, CA, USA), PE-conjugated CD106 (VCAM-1) (clone REA269, Miltenyi Biotec, Auburn, CA, USA), FITC-conjugated mouse anti-human CD44 (clone MEM-85, Invitrogen, Frederick, MD, USA), and PE-conjugated CD184 (CXCR4) (clone 12G5, Miltenyi Biotec, Auburn, CA, USA). The differences between forward-scatter and side-scatter signals in both cell types allowed reliable identification of each. Dead cells were excluded during acquisition and analysis (gate: intermediate forward-scatter and low side-scatter). FlowJo (v10.0, FlowJo, LLC, Ashland, OR, USA) was used for data analysis.

CD34+ cells were harvested by repeated pipetting after three days in the niches, washed in PBS (1X), and stained with monoclonal antibodies for FACS analysis: APC-conjugated mouse anti-human CD49d (clone 9F10, BD Pharmingen, San Jose, CA, USA), PE-conjugated mouse anti-human CD49e (clone IIA1, BD Pharmingen, San Jose, CA, USA), FITC-conjugated mouse anti-human CD44 (clone MEM-85, Invitrogen, Frederick, MD, USA), and APC-conjugated mouse anti-human CD54 (clone REA266, Miltenyi Biotec, Auburn, CA, USA). Reliable discrimination between the few MSC present and CD34+ cells was possible using their different forward-scatter and side-scatter signals. Dead cells were excluded during acquisition and analysis (gate: intermediate forward-scatter and low side-scatter). FlowJo (v10.0, FlowJo, LLC, Ashland, OR, USA) and Paint-A-Gate software (Pro v1.0, Becton Dickinson Biosciences, Sunnyvale, CA, USA) were used for data analysis.