Cd326 ep cam alexafluor 488

For sorting of cancer cells and CAFs, KPC tumors were processed as previously described (19 (link)). Cells were stained with anti-mouse CD45-AlexaFluor 647 (103124, BioLegend), CD326 (Ep-CAM)-AlexaFluor 488 (118212, BioLegend), CD31-AlexaFluor 647 (102416, Biolegend), CD140a (PDGFRα)-PE (135905, BioLegend), PDPN-APC/Cy7 (127418, Biolegend) and DAPI for 15 min. Cells were sorted on the FACSAria cell sorter (BD) for DAPI/CD45/CD31- EpCAM+ and DAPI/CD45/CD31/EpCAM- PDPN+ cell populations. For flow cytometry analysis of EdU-treated KPC tumors, KPC mice were administered 300 μg 5-Ethynyl-2-deoxyuridine (EdU) (61135-33-9, Santa Cruz) formulated in sterile saline twice a day for 3 days via intraperitoneal injection. EdU was detected using Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (C10634, Thermo Fisher Scientific). For flow cytometric analysis of IL-1R1 and myCAF/iCAF populations, antibodies employed were: anti-mouse CD31-PE/Cy7 (102418, Biolegend), CD45-PerCP/Cy5.5 (103132, Biolegend), CD326 (Ep-CAM)-AlexaFluor 488, PDPN-APC/Cy7, CD140a (PDGFRα)-PE, Ly6C-APC (128015, Biolegend), biotinylated CD121a (IL-1R, Type I/p80) (113503, Biolegend) and APC Streptavidin (405207, Biolegend).

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.