Multifunctional flow cytometry for EGFR-specific CTL

Recombinant epidermal growth factor, Zombie Aqua^™ Fixable Viability Kit, Alexa Fluor 488-EGFR, PerCP-Cy 5.5-CD3, PerCP-Cy 5.5-CD32 and PE-Cy5-CD86 flow antibodies were purchased from Biolegend (San Diego, CA). Cetuximab and panitumumab were purchased from the manufacturers, Bristol-Myers Squibb (New York, NY), and Amgen, (Thousand Oaks, California) respectively. FITC-CD69 and PE-Texas Red-CD8 flow antibody were purchased from Life Technologies (Grand Island, NY). PE-CD107a, PE-Cy5-CD137, PE-Cy7-CD16, APC-CD56, PE-CD32, APC-H7-HLA-A2 and FITC-CD80 flow antibodies were purchased from BD Biosciences (San Jose, CA). 12B6 antibody was produced by Dr. Ferrone (Harvard Medical School) and has been previously validated (14 (link)). PE-labeled HLA-A*0201-EGFR853-861 tetramer was provided by the NIH tetramer core facility and used for identification of EGFR-specific CTL (15 (link)). PE-Labeled HLA-A*02:01 HIV-tetramer was purchased from MBL International.