Anti cd3 fluorescein isothiocyanate

Peripheral blood samples (two mL) were collected from 51 GC patients and 50 healthy individuals in EDTA anticoagulant tubes. The lymphocyte subsets, including CD3+, CD4+, CD8+ T cells, CD19+ B cells, and CD16+ CD56+ NK cells, were quantified in cells/µL using a six-color flow cytometry assay. Human monoclonal antibodies, including anti-CD3-fluorescein isothiocyanate (FITC), anti-CD4-phycoerythrin (PE), anti-CD8-allophycocyanin (APC), anti-CD19-PE, anti-CD16-APC, and anti-CD56-PE (BD Multitest), were used according to the manufacturer’s instructions. The cell samples were analyzed on a BD Canto II flow cytometry system (BD Biosciences, East Rutherford, NJ, USA).

PBMCs were stimulated with phorbol myristate acetate (50 ng/mL) and ionomycin (1 µg/mL) in the presence of brefeldin A (10 µg/mL) for 6 h. Cells were stained with anti-CD3-fluorescein isothiocyanate (BD Pharmingen, San Jose, CA, USA; Clone UCHT1), anti-CD4–peridinin–chlorophyll–protein complex (BD Pharmingen, San Jose, CA, USA; Clone SK3), anti-CD8–phycoerythrin (BD Pharmingen, San Jose, CA, USA; Clone HIT8α) for 30 min in the dark at 4 ^∘C. Cells were then fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization (BD Biosciences, San Jose, CA, USA) and stained with anti-IL-9–allophycocyanin (R&D Systems, Minneapolis, MN, USA; Clone #623153). Flow cytometric analysis was performed using a BD LSR II System (BD Biosciences, San Jose, CA, USA).