Lm6090

For dual binding assays, 1×10⁵ MKN45 or 1×10⁵ PBMCs were incubated with 1 µg iRGD-anti-CD3 or control reagents for 30 min at 4°C, washed, and then incubated with a secondary mAb against His-tag conjugated to PE (GG11-8F3.5.1, Miltenyi Biotec GmbH). Expression of iRGD receptors on MGC803 and LOVO was measured by staining tumor cells with anti-αvβ3-FITC (LM6090, EMD Millipore), anti-αvβ5-FITC (P1F6, EMD Millipore), and anti-NRP-1-PE (AD5-17F6, Miltenyi Biotec GmbH). All samples tested were suspended in flow cytometry staining buffer (FACS) buffer and stained with the indicated antibodies for 30 min at 4°C in the dark, washed twice, and resuspended in FACS buffer before analysis. Flow cytometry data were collected on Accuri C6 (BD Bioscience, NJ, USA) and analyzed with FlowJo V.10.4 software. For cell sorting, activated T cells were incubated with anti-CD3-PC7 (5 µL/107 cells, MHCD0312; BD Bioscience), anti-CD4-FITC (5 µL/107 cells, MHCD0401-4; BD Bioscience), and anti-CD8-APC (5 µL/107 cells, MHCD0805; BD Bioscience) for 40 min at 4°C and washed. CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were negatively isolated on MoFlo XDP Cell Sorter (Beckman Coulter).