Mouse anti human cd9

Cells were suspended and washed three times with washing buffer composed of ice-cold D-PBS, 1% bovine serum albumin (BSA), 5 mM EDTA, and 25 mM Hepes before antibody incubation. hiPSCs were incubated with the primary antibody goat anti-human OCT3/4 (R&D Systems, Minneapolis, MN, USA), mouse anti-human SOX2 (R&D Systems), goat anti-human NANOG (R&D Systems), mouse anti-human PODPCALYXIN (R&D Systems), mouse anti-human SSEA4 (R&D Systems), or mouse anti-human CD9 (R&D Systems) for 30 min at 4°C. After three subsequent washes with the buffer, cells were incubated with secondary antibody Alexa Fluor 546 donkey anti-mouse (Thermo Fisher Scientific) or fluorescein isothiocyanate (FITC) donkey anti-goat (Santa Cruz Biotechnology, Santa Cruz, CA) for another 30 min at 4°C. For analysis of MC-Chs and NCC-Chs, cells were incubated with phycoerythrin-conjugated mouse anti-human CD44 or CD151 (BD) for 30 min at 4°C. Fluorescent cells were then detected by the Attune NxT Flow Cytometer (Life Technologies, Carlsbad, CA), and the resulting flow cytometry data were analyzed by FlowJo (Tree Star, Ashland, OR), following the manufacturer’s instructions.