Apc conjugated anti cd44

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.

APC‐conjugated anti‐CD44 (#103011, 1:200; Biolegend, San Diego, CA, USA), phycoerythrin (PE)‐conjugated anti‐glial fibrillary acidic protein (GFAP; #FCMAB257P, 1:40; Millipore, Burlington, MA, USA), PE‐conjugated anti‐microtubule‐associated protein 2 (MAP2; #FCMAB318PE, 20 μl per test; Millipore), and control IgG (#555749, 1:40; BD Biosciences) antibodies were used for FACS. To obtain CD44^High or CD44^Low cells, patient‐derived primary tumor cells or transplanted murine primary tumor cells stained with 1:200 dilutions of APC‐conjugated anti‐CD44 antibody were subjected to FACS. For cell cycle analyses, cells were incubated with 20 μm BrdU (#B5002; Sigma‐Aldrich, St Louis, MO, USA) for 3 days, followed by incubation with PE‐conjugated anti‐BrdU antibody (#339811, 1:40; Biolegend). Apoptotic cell death was measured using an APC‐ (#ab236215; Abcam, Cambridge, UK) or FITC‐conjugated (#ab14085; Abcam) Annexin V Apoptosis Detection Kit I.