Facsaria cell sorter system

FACS-based sorting and analysis of marker ZIP4⁺ cells was conducted using the BD FACSAria cell sorter system (Becton-Dickinson, Franklin Lakes, NJ, USA) and BD LSR Fortessa Analyser (BD Biosciences, San Jose, CA, USA), and data analyzed by FlowJo V10 (BD Biosciences, San Jose, CA, USA) as we described in [17 (link)].

Human PE01, PE04, PEA1, PEA2, and Kuramochi cells were detached and labeled by the ZIP4 antibody (AF7315; R&D Systems, 1:50) at room temperature for 2 h, followed by incubation with a APC-labeled donkey anti-goat IgG secondary antibody (F0108; R&D Systems, 1:500). ALDH activity was measured using the ALDEFLOUR Kit (STEMCELL Technologies, Vancouver, Canada) as we described previously [15 (link)] and detected using the green fluorescence channel (520–540 nm). FACS-based sorting and analysis of markers ZIP4 and ALDH were conducted using the BD FACSAria cell sorter system (Becton-Dickinson, Franklin Lakes, NJ, USA) and BD LSR Fortessa Analyzer (BD Biosciences, San Jose, CA, USA), and data analyzed by FlowJo V10 (BD Biosciences, San Jose, CA, USA). For the self-renewal assay, FACS-sorted ZIP4⁺ cells (100% pure) were collected and seeded into triple wells (1 × 10⁴ cells per well), cultured under differentiation conditions (DMEM medium with 10% FBS) for 48 h, trypsinized using 0.25% trypsin and washed twice in PBS, then cells were relabeled with ZIP4/APC antibodies before the renew/differentiation assays.