Anti human cd107a apc

Vγ9Vδ2 T cells were stimulated with 5 μg/mL plate-bound anti-CD3 antibodies and 1 μg/mL soluble anti-CD28 antibodies (free mAbs) for 6 h in the presence of 5 μg/mL Brefeldin A. Then, the cells were stained with anti-human CD3-APC-H7 (BD Biosciences, clone: SK7), anti-human TCR Vδ2-PerCP (BioLegend, clone: B6), and anti-human CD107a-APC (BD Biosciences, clone: H4A3) antibodies. After staining for surface markers, the cells were fixed and permeabilized using Lysing Solution (BD Biosciences) and Permeabilizing Solution (BD Biosciences), respectively. Subsequently, the cells were stained with anti-human IFN-γ-PE-Cy7 (BD Biosciences, clone: B27), anti-human TNF-α-PE (BD Biosciences, clone: MAb11), and their corresponding isotype controls (APC-conjugated mouse IgG1, κ isotype control (clone: MOPC-21); PE-Cy™7-conjugated mouse IgG1, κ isotype control RUO (clone: MOPC-21); and PE-conjugated mouse IgG1, κ isotype control (clone: MOPC-21), all from BD Biosciences). Then, the cells were analyzed with a BD FACS Verse, and the data were analyzed with FlowJo. In addition, cell cycle, cell proliferation (Ki-67, BioLegend, clone: Ki-67), cell differentiation (CD45RA, BioLegend, clone: HI100), CD27 (BioLegend, clone: O323), mitochondrial, and costimulatory molecule (CD80, BioLegend, clone: 2D10; CD86, BioLegend, clone: BU63) analyses were performed following standard protocols.

1 × 10⁵ cells of PC-3 were pre-stained with Cell Trace CFSE (Invitrogen) and then co-cultured with NK-92MI cells at 10:1 effector: target (E: T) ratio. After 4 h of co-incubation, cells were harvested and stained with 7-AAD (Invitrogen) for the discrimination of live and dead cells. To assess the degranulation of NK cells, co-cultured cells were stained with anti-human CD107a-APC (BD Biosciences) for 1 h at 4 °C, then analyzed on a flow cytometer (BD Bioscience).