Facsariatm 2 cell sorter

Foxp3⁺ Tregs were purified from tumors of Foxp3GFP mice. Tumor single-cell suspensions were generated as described previously. CD4+ T cells were enriched by Ficoll gradient (Sigma-Aldrich), before sorting for GFP expression on a FACSAriaTM II Cell Sorter (BD Biosciences). Dead cells and doublets were excluded on the basis of forward and side scatter and Fixable Viability Dye eFluor 506. Purity of flow-sorted populations was above 90%.

The 3D collagen-fibrin gel-based killing assay has been previously described in depth¹⁹ . For the isolation of intratumoral CD8⁺ T cells, B16^WT and B16^IDO tumors were excised on day 15 and dissected into single-cell suspensions, as described above. Tumor-isolated CD8⁺ T cells were enriched by Ficoll gradient (Sigma-Aldrich) before sorting as (CD45⁺TCRβ⁺CD8⁺) on a FACSAriaTM II Cell Sorter (BD Biosciences). In brief, 0.1 × 10⁵ viable B16-F10 target cells were co-embedded into collagen–fibrin gels with 1 × 10⁵ FACS-sorted effector CD8⁺ T cells in a 10:1 ratio (effector:target). CD8⁺ T cells isolated from the spleen of Pmel-1/gp100-specific TCR transgenic mice were used as a positive control. After 48 h of co-culture, collagen-fibrin gels were dissolved and target cells were diluted and plated into 6-well plates for a colony formation assay. After 7 days, cells were fixed using 3.7% formaldehyde and stained with 2% methylene blue before colony counting.

High-resolution ultrasound imaging was used to identify a dominant mass that met enrollment criteria. For sorting of Fzd4⁺ CAFs and Fzd4^- CAFs, tumor tissues from KPC (Kras^{LSL-‍G12D/+}; Trp53^R172H/+; Pdx1-‍Cre) mice were minced and enzymatically digested in PBS supplemented with 5% FBS, 1 mg/ml Type VIII Collagenase (Sigma, #C2139), 2 mg/ml Dispase II (Sigma, #4942078001), 1 mg/ml Trypsin Inhibitor (Sigma, #T6522) and 1 unit/ml DNase I (NEB, # M0303S) for 45 min at 37 °C with agitation. Cell digestion of both tumor and adjacent normal samples were strained through 100 μm cell strainer and resuspended in PBS supplemented with 0.5% FBS. Cells were stained with anti-Fzd4 (R&D Systems, #MAB194), and rabbit anti-rat IgG-PE (Solarbio, #K0032R-PE) was used as the secondary antibody. Cells were subsequently stained with anti-CD45-AlexaFluor 647 (BioLegend, #103124), anti-CD326 (Ep-CAM)-AlexaFluor 488 (BioLegend, #118212), anti-CD31-AlexaFluor 647 (BioLegend, #102416) and anti-PDPN-APC/Cy7 (BioLegend, #127418). Cells were sorted on the FACSAria^TM II cell sorter (BD) for CD45/CD31/EpCAM^- PDPN⁺ Fzd4⁺ and CD45/CD31/EpCAM^- PDPN⁺ Fzd4^- cell populations.

iMPCs were trypsinized, washed and incubated with the following antibodies: FITC Mouse Anti-Human CD31, CD34, CD45, CD73, and CD90 (BD Biosciences, Franklin Lakes, NJ). Cells were then analyzed by flow cytometry (BD FACS Aria^TM II cell sorter; BD Biosciences) to assess the expression of these cell surface epitopes.

An Annexin V–fluorescein isothiocyanate (FITC) apoptosis detection kit (Neobioscience, Inc., Shenzhen, China) was used to assess the apoptosis. HK-2 cells were seeded in 6-well plates. The adhered cells were exposed to different media for 24 h or 72 h and then detached with trypsin. Subsequently, the cells were resuspended in a binding buffer (195 mL) and stained with Annexin V–FITC (5 μL) and propidium iodide (PI; 10 μL) at room temperature for 10 min in the dark. Apoptotic cells were determined using a flow cytometer (BD FACSAria^TM II cell sorter). Annexin V–FITC-positive and PI-negative cells were considered apoptosis.

The whole pancreases were dissected from double transgenic adult zebrafish [Tg(ins:GFP, ela:mCherry), Tg(ins:GFP, gcga:mCherry), and Tg(ins:GFP, sst:mCherry)] and fixed using 4% formaldehyde (#F1635, Sigma-Aldrich) in 1xPBS [137 mM NaCl (#S/3161/60, Fisher Chemical), 2.7 mM KCl (#2676.298, VWR), 10 mM NaHPO4 (#1.06342.0250, Merk), and 1.8 mM KH2PO4 (#1.06585.1000, Merk)]. Cells were dissociated, on ice, using a 15 mL Dounce homogenizer in 1 mL of ice-cold sort buffer [1% EDTA (#20301.290, VWR), 2 mM HEPES (#83264, Sigma-Aldrich) pH 7.0 in 1xPBS), and then passed through a 40-μm cell strainer. Immediately following dissociation, the mCherry and GFP fluorescence were analysed on a BD FACS-ARIATM II cell sorter (BD Biosciences).