Cd8 bv570 clone rpa t8

Previously stimulated samples were thawed and washed in FACS buffer (PBS, 5% FBS v/v, 0.05% sodium azide w/v) and stained for 30 minutes at 4 °C in the dark, with a cocktail of surface marker antibodies: CD3-BV650 (clone SK7), CD4-BV605 (clone S3.5), CCR7-PECF594 (clone 150503), CD45RO-APC-H7 (clone UCHL1), (all BD Bioscience) and CD8-BV570 (clone RPA-T8; BioLegend). Samples were then permeabilised using the Cytoperm/Cytofix (BD Pharmingen) solution for 20 mins. After permeabilisation, cells were stained for 30 minutes at room temperature in the dark with a cocktail of intracellular cytokine antibodies: TNFα-PE-Cy7 (clone Mab11), IFNγ-V450 (clone B27), IL-2-FITC (clone MQ1-17H12), (BD Bioscience), IL-4-PE (clone 3010.211; FastImmune), IL-5-PE (clone TRFK5), IL-13-PE (JES10-5A2) and IL-8-APC (clone E8A1), (BioLegend). IL4, 5 and 13 were collected in one PE channel. After staining, samples were washed and stored at 4 °C in the dark to be acquired within 24 hours using BD LSRII Flow Cytometer and acquiring a minimum of 200,00 events.

Simulations were performed as described [38 (link)]. In short, cryopreserved PBMC were thawed and rested overnight in R10/RPMI 1640 (BioWhittaker, Walkersville, MD), 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin] with 50 U/ml Benzonase (Novagen, Madison, WI) in a 37°C/5% CO₂ incubator. The following morning, cells were stimulated with peptide pools (2 μg/ml) in the presence of GogiPlug (10 μg/ml; BD Biosciences, San Jose, California) for 6 h. Negative controls received an equal concentration of DMSO instead of peptides. Subsequently, intracellular cytokine staining (ICS) was performed as described [39 (link)]. The following monoclonal antibodies were used: CD4-BV421 (clone OKT4; BioLegend), CD8-BV570 (clone RPA-T8; BioLegend), CD69-ECD (clone TP1.55.3; Beckman Coulter), CD3-Cy7APC (clone SP34.2; BD Biosciences), IFN-γ-APC (clone B27; BD Biosciences), IL-2-PE (clone MQ1-17H12; BD Biosciences), and TNF-FITC (clone Mab11; BD Biosciences). Aqua LIVE/DEAD kit (Invitrogen, Carlsbad, CA) was used to exclude dead cells. All antibodies were previously titrated to determine the optimal concentration. Samples were acquired on an LSR II flow cytometer and analyzed using FlowJo version 9.8 (Treestar, Inc., Ashland, OR).