Pe cy7 conjugated anti cd200 ox 104

Flow cytometry was performed according to a previously published method (Hayashi et al., 2018 (link)). Briefly, hiPSC-derived SEAMs were dissociated using TrypLE Express (Thermo Fisher Scientific) and re-suspended in ice-cold keratinocyte culture medium (KCM) (Hayashi et al., 2016 (link), 2017 (link), 2018 (link)) supplemented with 5% FBS (Japan Bio Serum, Hiroshima, Japan). Harvested cells were then stained with 5 μg/1×10⁶ cells of mouse anti-ABCB5 mAb (clone 3C2-1D12) (Ksander et al., 2014 (link); Wilson et al., 2014 (link); Lutz et al., 2016 (link); Schatton et al., 2015 (link)) for 30 min on ice. Following three washes with PBS they were then stained with 1 μg/1×10⁶ cells of Alexa Fluor 647-conjugated secondary antibody (Thermo Fisher Scientific) for 30 min on ice. Cells were subsequently stained with 0.5 μg/1×10⁶ cells of PE-conjugated anti-CD104 (ITGB4; 58XB4, Biolegend, San Diego, CA), 0.375 μg/1×10⁶ cells of FITC-conjugated anti-SSEA-4 (MC813-70, Biolegend), and 0.25 μg/1×10⁶ cells of PE-Cy7-conjugated anti-CD200 (OX-104, BD Biosciences, San Diego, CA) antibodies for 1 hr on ice, followed by three washes with PBS. Cell sorting was performed with a FACSAria II instrument (BD Biosciences), and the data analyzed using BD FACSDiva Software v8.0.3 (BD Biosciences).