Cd326 pe vio770

Digested tumor or adjacent normal cells were centrifuged and resuspended at 5 × 10⁵–2 × 10⁶ cells/100 µL FACs buffer (Miltenyi, Bergisch Gladbach, Germany). Cells were divided into no antibody control, viability analysis panel (Propidium iodide, Hoeschst33342, DRAQ7), IgG control (REA-VioBlue, REA-FITC, REA-PE, REA-APC, REA-PE-Vio770), staining panel 1 (1:50 CD31-VioBlue (Miltenyi 130-117-227), 1:11 CA9-PE (Miltenyi 130-110-057), 1:10 PDGFRβ-APC (Miltenyi 130-105-322), 1:10 PDGFRα-APC (Miltenyi 130-115-239), 1:50 CD326-PE-Vio770 (Miltenyi 130-111-002), 1:50 CD45-FITC (Miltenyi 130-110-631), Propidium Iodide), or staining panel 2 (1:50 CD10-VioBlue (Miltenyi 130-114-509), 1:11 CA9-PE, 1:50 CD105-PE-Vio770 (Miltenyi 130-112-167), CD184-APC (Miltenyi 130-098-357), 1:50 CD326-PE-Vio770, Propidium Iodide), and incubated in the dark at 4 °C for 15 min. Cells were washed two times with flow buffer and analyzed using Miltenyi AutoMACs flow cytometer. Side and forward scatter gating were determined using viability analysis panel to identify cells vs. debris. Doublets were excluded using SSR-H versus SSR-L. Positive gating for each marker was determined using IgG control. Compensation was performed using the MACS Comp Bead kit, anti-REA (Miltenyi 130-104-693).