Live dead fixable yellow dead cell stain kit

Single-cell suspensions were generated from biopsies, and FACS was performed on ice for 30 minutes with the following antibodies: Invitrogen Live/Dead Yellow (catalog L34959, Thermo Fisher Scientific, LIVE/DEAD™ Fixable Yellow Dead Cell Stain Kit for 405-nm excitation), human PDPN PE (catalog 337003), human CD45 APC-Cy7 (catalog 304014), and human E-cadherin (E-cad) APC (catalog 324108) (all purchased from BioLegend Inc.). The 2 populations of interest were sorted by MoFlo machine under 2-way sorting settings with representative sorting chart shown (Figure 3A). Live singlet events were preselected by a Live/Dead+ FSC-A/FSC-H diagonal gate. The PDPN^hi population was defined and sorted as live CD45-PDPN^hiE-cad^hi events, whereas the PDPN^lo population was defined as live CD45-PDPN^loE-cad^hi events. Sorting typically lasted around 30–45 minutes, with frequent agitation to collect the droplets attaching to the collection tube wall. Three biopsies collected from the distal esophagus typically yielded approximately 2500–10,000 epithelial cells in each subpopulation; they were subsequently collected into RPMI-1640 media supplemented with 10% FBS and pelleted/centrifuged for immediate RNA isolation procedures using RNeasy Micro Kit (Qiagen).