Macs ld depletion columns

Human NSCLC cell lines A549 and PC9 were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen, Carlsbad, CA, USA) in an incubator with 5% CO 2 at 37 °C. CD133 + and CD133 -populations of the A549 and PC9 cell lines were sorted by using an anti-CD133-FITC antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) with flow cytometry (FACSCALIBUR; BD Biosciences, Franklin Lakes, NJ, USA). γδ T cells were expanded ex vivo by using blood samples obtained from healthy donors (n = 10; informed consent was obtained from all participants). In brief, peripheral blood mononuclear cells (PBMCs) were separated by using the Ficoll reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. To induce the generation of γδ T cells, PBMCs were cultured in DMEM supplemented with 10% FBS, 3 μM BrHPP (Innate Pharma, Marseille, France), and 400 IU/mL IL-2 (R&D Systems, Minneapolis, MN, USA) for 2 weeks [20] . To purify the γδ T cells, the abovementioned cells were negatively selected by using an anti-αβ-TCR antibody (BioLegend, San Diego, CA, USA) and MACS LD depletion columns (Miltenyi Biotec) [21] . γδ T cells were washed 3 times before co-culture with A549 and PC9 cells.