Anti cd45

Previously fixed samples were centrifuged at 400× g for 10 min to remove PBS. Cells were resuspended in 100 μL of 1% bovine serum albumin (BSA) (Sigma, St. Louis, MO, USA) and incubated for 10 min. Samples were centrifuged again to remove BSA before being resuspended in 90 μL of Running Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany). A primary antibody staining cocktail of the following biotinylated antibodies was added and incubated for 15 min at 4 °C: 10 μL of anti-EpCAM (R&D system, Minneapolis, MN, USA), 10 μL of anti-CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany), 5 μL of anti-EGFR (RayBiotech, Peachtree Corners, GA, USA) and 20 μL anti-CD45 (BioRad, Hercules, CA, USA). After 15 min, the reaction was quenched with 1 mL of Running Buffer before being centrifuged. The supernatant was discarded and the following solutions were added before incubating an additional 10 min at 4 °C: 90 μL of Inside Stain (Miltenyi Biotec, Bergisch Gladbach, Germany), 2.5 μL of strepavidin Alexa Fluor 488 conjugated (Invitrogen, Waltham, MA, USA), 10 μL of goat anti-mouse IgG2a AF647 (Invitrogen, Waltham, MA, USA), 10 μL of PE anti-pan cytokeratin (Abcam, Cambridge, UK) and 2.5 μL of DAPI (Invitrogen, Waltham, MA, USA). After incubation, the reaction was quenched with 1 mL of Running Buffer and centrifuged.