Cd3 percp clone sk7

To assess CD8⁺ T-cell degranulation, purified CD8⁺ T cells were incubated in the presence or absence of P815 target cells at a 1:1 ratio for 30, 60, 90, 120, and 180 min at 37 °C. Cells were then stained using the following fluorescently labeled antibodies: anti-CD107a-PE (clone H4A3), CD56-APC (clone NCAM16.2), CD8-FITC (clone SK1), and CD3-PerCP (clone SK7), all of which were from BD Biosciences (San Jose, CA). Data were collected using a Cyto-Flex-S cytometer (Beckman). CD3⁺CD8⁺ cells were gated and assessed for surface expression of CD107a.

On 96-well flat-bottom plates, 2 × 10⁵ of PBMCs were seeded (Greiner Bio-One, Kremsmünster, Austria) and cultured for 24 h at 37 °C in a humidified atmosphere with 5% CO₂, in the presence of Se-Le-30 (100 µg/mL) and with an equivalent amount of medium and water for injection as controls. After incubation, cells were harvested, washed in 2 mL of PBS, resuspended in 100 µL of Zombie Violet™ (BioLegend, San Diego, CA, USA) stock solution at a ratio of 1:400, incubated for 20 min at room temperature in the dark, then washed with 2 mL of Stain Buffer and labeled with mouse anti-human CD3 Ab (CD3-PerCP, Clone SK7, BD Biosciences, San Jose, CA, USA) in 100 µL of Stain Buffer for 15 min. Next, cells were washed in 2 mL of Stain Buffer, resuspended in 100 µL of PBS wit 0.01% sodium azide, and acquired with DxFlex flow cytometer. For each variant, the percentage of CD3⁺ T lymphocytes positive for Zombie Violet dye was recorded and compared to control cultures.

The thawed PBL, allowed to rest overnight as described above, were used for an in vitro proliferation assay using the fluorescent cell proliferation indicator CytoTell green (AAT Bioquest; LuBioScience), according to the manufactures protocol (20 min incubation at room temperature in 1:600 diluted dye). Cells (150′000 in 270 μl per well) were plated in duplicates in a 96 U-bottom well plate (TTP, Switzerland) either in control medium or in stimulation medium with 2.5 μg/ml pokeweed mitogen or in stimulation medium with 0.1% phytohaemagglutinin (PHA-M, Sigma-Aldrich, Switzerland). The plates were incubated at 37 °C and 5% CO₂ in humid atmosphere. Proliferative responses of the stimulated PBL and the controls were measured after 7 days of incubation by flow cytometry using the intracellular dye dilution method and following surface marker antibodies: CD3-PerCP (clone SK7, BD biosciences), CD4-APC (clone RPA-T4, BD bioscience), CD8-PE (clone RPA-T8, BD biosciences). Cells were incubated with 5 μl of antibodies for 15 min at room temperature, washed and resuspended in 200 μl PBS. Cell fluorescence was assessed with FACScalibur flow cytometer and data were analyzed using the proliferation tool of the FlowJo, 9.0 software.