Cd8 pe clone rpa t8

The CD4/CD8 ratios in peripheral blood were evaluated by flow cytometry using the fluorochrome labeled monoclonal antibodies: CD3-FITC (clone SP34; BD Pharmingen); CD4-PerCP (clone L200; BD Pharmingen); and CD8-PE (clone RPA-T8; BD Pharmingen). Antibodies, diluted as per manufacturer's instructions, were added to 100 μL of EDTA-whole blood and incubated for 30 min at room temperature. Erythrocytes were then lysed by BD FACS lysing solution (Becton, Dickinson). Cells were washed once with 1 × PBS and resuspended in 1 × PBS containing 1% paraformaldehyde. Unstained and single color control samples were acquired to calculate the compensation matrix. Gating was performed on mononuclear cells and then on CD3⁺CD4⁺ or CD3⁺CD8⁺ subpopulations. The flow cytometry analysis was performed on a BD FACSCalibur (Becton Dickinson Biosciences, San Jose, CA) and analyzed using CellQuest software (Becton Dickinson).

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.