EGFR Expression Quantification Protocol

EGFR expression was measured with immunolabeling using mouse monoclonal antibody mAb 528 (isolated from the supernatant of the hybridoma ATCC HB-8509), with a slightly different binding site [27 (link)] and cetuximab (Merck). Alexa Fluor 647-conjugated secondary antibodies (goat-anti-mouse (GAMIG) and goat-anti-human (GAHIG), Invitrogen, Carlsbad, CA, USA) were used. The 10⁶ cells were labeled with 10 µg/mL of antibodies for 10 min on ice in PBS supplemented with 5 mM glucose. After each incubation, cells were washed twice by centrifugation at 400× g. At least 10,000 cells per sample were analyzed with an Attune NxT Acoustic Focusing Cytometer (Applied Biosystems, Life Technologies, Carlsbad, CA, USA).