Anti cd31 apc

Experimental animals, 20/group, were each implanted in the brain with 1 × 10⁵ U87-eGFP-PLuc, NCH644-eGFP-PLuc or NCH421k-eGFP-PLuc cells. Tumors were extracted and mechanically disaggregated using a scalpel. Single cells were separated from tissue clumps using a cell strainer and incubated with red blood cells lysis buffer during 5 minutes. Individual eGFP expressing tumor cells positive for pericytic and endothelial cell markers were isolated using a fluorescence-activated cell sorter (FACS). Pericyte-like cells were sorted from total cells by selecting eGFP+/CD146+/CD248+ cells with anti CD146-VioBlue (Miltenyi Biotec) and anti CD248-Alexa Fluor 647 (clone B1/35 Beckton Dickinson). Endothelial-like cells were sorted by selecting eGFP+/CD31+/CD105+ cells with anti CD31-APC (Miltenyi Biotec) and CD105-VioBlue (Miltenyi Biotec). Selected positive cells of either type were grown in non-adherent plates with tumorsphere culture media.

The phenotype of hESC-MSCs was characterized using flow cytometry and the following monoclonal antibodies: anti-CD31-APC (Miltenyi Biotec), anti-CD44-FITC (BD Pharmingen), anti-CD45-FITC (BD Pharmingen), anti-CD73-APC (Miltenyi Biotec), anti-CD90-FITC (BD Pharmingen), anti-CD105-PE (eBioscience), HLA-ABC-APC (BD Pharmingen), and HLA-DR-FITC (BD Pharmingen). Briefly, the cells were dissociated and suspended in FACS buffer (1x PBS/0.5% BSA) and nonspecific binding blocked with FcR blocking agent (Miltenyi Biotec) for 10 minutes at 4°C. For labeling cell surface antigens, the cells were incubated with the abovementioned fluorescent conjugated antibodies for 10 minutes at 4°C. After antibody labeling, data was acquired using Dako Cytomation CyAn ADP and analyzed using FlowJo v7.6.5 (Tree Star).