Anti cd3 buv805

The production of proangiogenic factors by NK cells was studied in 8 HCC patients (paired LINK and TINK) after overnight stimulation with IL-12 and IL-18 (5 ng/mL) in the presence of 10 µg/mL of brefeldin A (BFA). Surface staining with anti-CD3-BUV805, anti-CD56-BUV805, and anti-CD49a-BV510 (BD Bioscience, Franklin Lakes, NJ, USA) was performed. Cells were then fixed and permeabilized with Fixation Permeabilization Concentrate and Diluent and Permeabilization Buffer (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions and stained with Eomes-PE-Cy7 (Invitrogen), Angiopoietin 1 (ANGPT1)-Alexa Fluor750 (Bioss Antibodies, Boston, MA, USA), CXCL10/IP-10-Alexa Fluor 700 (R&D System, Minneapolis, MN, USA), Osteopontin-eFluor660 (Thermo Fisher Scientific, Waltham, MA, USA), IL-8-PerCp-eFluor710 (Thermo Fisher Scientific, Waltham, MA, USA), MMP-9-Alexa Fluor 488 (Abcam, Cambridge, UK), Placental growth Factor (PlGF)-Alexa Fluor594 (Bioss Antibodies, Boston, MA, USA), VEGF-PE (R&D system, Minneapolis, MN, USA), and Angiogenin-Alexa Fluor405 (Novus Biotechne, R&D System, Minneapolis, MN, USA). Analysis was performed on an 18-fluorescence flow cytometer (FACS Fortessa, Becton Dickinson (BD) Immunocytometry System, CA, USA). Data are expressed as the percentage of cytokine-positive cells in the CD49a+Eomes+ NK cell subset.

For analysis of surface markers, T cells were collected at indicated time points after activation with CD3/CD28 Dynabeads (11131D, Thermo Fisher). For analysis of CD11b expression on T cells, activated T cells were collected at day 8, washed in Flow Cytometry Staining Buffer (00-4222-26, Thermo Fisher) and incubated with anti-CD11b biotin antibody (553309, BD Biosciences; 1:100) for 30 min at 4 °C. Cells where then washed and incubated with anti-CD3 BUV805 (612894, BD Biosciences; 1:100), anti-CD4 BUV395 (563550, BD Biosciences; 1:200) anti-CD8 APC/H7 (566855, BD Biosciences; 1:200) and streptavidin PE (12-4317-87, Thermo Fisher; 1:200) for 30 min at 4 °C in the dark. After washing, cells were resuspended in 200 µL staining buffer containing DAPI (1:10,000) (D1306, Invitrogen).
For STn surface staining, cells were incubated with anti-STn primary antibody (ab115957, Abcam; 1:100) for 30 min at 4 °C. Cells were then washed and incubated with an anti-mouse IgG-FITC secondary antibody (406001, Biolegend; 1:200) for 30 min at 4 °C in the dark. After a final wash step, cells were resuspended in 200 µL of 2 % BSA in PBS with DAPI (1:10,000). Data were collected on a FACSymphony flow cytometer and analyzed using FlowJo (BD Biosciences).