Apc conjugated anti cd34

Mouse kidney tissue was diced and digested with Collagenase-I at 37°C for 1 hour. The suspension was filtered through a 40 um strainer to remove cell pellets and washed in PBS to generate a single cell suspension after lysis of red blood cells (BD, USA). The cells were washed twice with PBS. The third generation of hAD-MSCs were trypsinized and washed twice with PBS. The cells were then incubated with the following fluorescent antibodies and corresponding isotype controls (Cat.Number: 400605, 400611 and 400507, Biolegend, USA) for 30 minutes shielded from light at room temperature: FITC-conjugated anti-CD45, APC-conjugated anti-CD11b, PE-Cy7-conjugated anti-CD3, PE-conjugated anti-F4/80, APC-conjugated anti-CD34, PE-conjugated anti-CD31, FITC-conjugated anti-CD90, APC-conjugated anti-CD44 and PE-conjugated anti-105 (Biolegend, USA). The positive cells were sorted using a BD FACS and the data were analysed using the FlowJo v7.6.3 software.

The expression of cell surface molecules was analysed by flow cytometry (FACSAria; Becton Dickinson, Franklin Lakes, NJ, USA). On day 14, a fraction of the HPCs within the ES-sacs were stained with CD34 antibody for 30 minutes on ice. Nonadherent cells on day 20 of culture were prepared in PBS containing 3% FBS (staining medium) and stained with combinations of antibodies for 30 minutes on ice. All samples were then washed with staining medium and analysed by flow cytometry. The following antibodies were used: allophycocyanin (APC)-conjugated anti-CD34 (Biolegend, San Diego, CA, USA), APC-conjugated anti-CD41a (integrin αIIb subunit, Biolegend), phycoerythrin (PE)-conjugated anti-CD41a (Biolegend), PE-conjugated anti-CD42b (Glycoprotein Ibα, Biolegend), PE-conjugated anti-CD71 (BD Pharmingen, San Diego, CA, USA), and APC-conjugated anti-CD235 (Glycophorin A, Biolegend).