Aria 2 sorter

Isolation of primary PDAC cells from mouse pancreatic tumors was performed as previously described with minor modifications (Chen et al., 2018 (link); Zheng et al., 2015 (link)). Fresh tumor tissues were minced with sterilized lancets, digested with collagenase IV (17104019, Gibco, 4 mg/mL)/dispase II (17105041, Gibco, 4 mg/mL)/RPMI at 37 °C for 0.5 hour, filtered by 70 μm cell strainers to generate single cell suspension and resuspended in RPMI/2%FBS. The subsequent single-cell suspension was stained with CD140a(PDGFRα)-PE (135905; BioLegend) for fibroblast sorting, and CD326(EpCAM)-AlexaFluor-488 (118210; BioLegend) for cancer cell sorting, as previously described (Chen et al., 2021 (link)). Samples were filtered through a 40 μm mesh and then sorted with Aria II sorter (BD Biosciences) at the South Campus Flow Cytometry Core Laboratory of MDACC. Cells were cultured in RPMI medium containing 20% FBS and 1% penicillin-streptomycin-amphotericin B (PSA) antibiotic mixture. Human cell lines, such as Panc1, BxPC3, PSN1, CAPAN1, T3M4, HPNE (human pancreatic epithelial cells), and BJ (fibroblasts), were cultured in RPMI with 10% FBS and 1% penicillin–streptomycin (PS). All cell lines were from American Type Culture Collection (ATCC) except for T3M4 (Cell Bank, RIKEN BioResource Center). Cells were routinely tested to be negative for mycoplasma.