For flow cytometric analysis, patient blood was collected, as described above. Red blood cell (RBC) lysis was performed by a 1-min incubation in 0.2% NaCl, followed by addition of the equivalent volume of 1.6% NaCl. Cells were washed and resuspended in FACS buffer [phosphate-buffered saline (PBS), 1.0 mM EDTA, and 5% fetal bovine serum (FBS)]. Cells were incubated in PBS containing LIVE/DEAD Fixable Aqua (1:500; Invitrogen) with Fc Receptor Binding Inhibitor (1:200; eBioscience). Cells were then incubated in FACS buffer for 30 min with CD45-APC (1:25; Thermo Fisher Scientific), CD11c-APCeF780 (1:100; Thermo Fisher Scientific), CD14-BV785 (1:100; BioLegend), CD163-PECy7 (1:100; BioLegend), EPCAM-FITC (1:100; Abcam), or cytokeratin-PE (1:500; Abcam). A BD LSRFortessa cell analyzer was used for analyses. A gating scheme established with single-color controls is provided in fig. S10. Data reflect analyses from n = 3 patients with PDAC.

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