Fitc conjugated mouse anti human cd31

Fluorescence-activated cell sorting (FACS) analysis was used to detect cell surface markers. Cells were stained for 60 min at 48 °C, then fixed with 2% paraformaldehyde. The surface markers investigated were FITC-conjugated mouse anti-human UEA, FITC-conjugated mouse anti-human CD31, and Alex-488-conjugated mouse anti-human c-kit (all from BD PharMingen, Franklin Lakes, NJ, USA). Isotype-identical antibodies served as negative controls. Analysis involved the use of FACS Calibur (Becton, Dickinson and Company (BD), Franklin Lakes, NJ, USA) and Cell Quest software.

Endothelial cells and mesenchymal cells were derived as described previously^{15 (link)}. iPSC-NSC were derived as described previously^{31 (link)}. Gene expression was evaluated using qPCR and flow cytometry. Following antibodies were used for flow cytometric analysis: PE-conjugated mouse anti-human CD140b (PDGFRB; 558821, BD Biosciences); APC-conjugated anti-human CD166 (ALCAM; 130-119-809, Miltenyi Biotec); anti- human CD105, human CD73, and human CD90 (MSC phenotyping kit; 130-095-198, Miltenyi Biotec); FITC-conjugated mouse anti-human CD31 (557508, BD Biosciences); PE-conjugated mouse anti-human CD144 (561714, BD Biosciences).

For the characterization of epithelial-like cells compared with HERS/ERM, fluorescence-activated cell sorting (FACS) was performed. The cells were detached and washed with DPBS supplemented with 2% FBS and then fixed with 4% paraformaldehyde at RT for 10 min. After washing with DPBS, 10,000 cells were incubated with fluorescently conjugated antibodies for 20 min at 4 °C. The following antibodies were used: FITC-conjugated mouse anti-human CD31, HLA-DR, HLA-I, PE-conjugated mouse anti-human CD10, CD29, and APC-conjugated mouse anti-human CD45 (all from BD Pharmingen, San Diego, CA, USA). The fluorescence intensity was measured by a FACS Calibur, and data were analyzed with FlowJo software (Tree Star, San Carlos, CA, USA).