Cd4 percp cy5.5 mab clone 74 12 4

PBMC in cRPMI were seeded at 1 × 10⁶ cells/well in 96-well round bottom plates (Nunc, Thermo Fisher Scientific) and stimulated with PRRSV-1 Olot/91 at a MOI = 0.1, in triplicate wells. PBMC incubated in triplicate wells with an equivalent volume of mock virus supernatant served as a negative control. After 18 h at 37 °C, 5% CO₂, BD GolgiPlug (1:1000; BD Biosciences) was added and cells further incubated for 6 h at 37 °C. PBMC were then surface stained with Zombie NIR fixable viability stain (BioLegend), CD3-FITC mAb (clone BB23-8E6-8C8, BD Biosciences), CD4-PerCP-Cy5.5 mAb (clone 74-12-4, BD Bioscience) and CD8α-PE mAb (clone 76-2-11, BD Bioscience), and intracytoplasmically stained with IFN-γ-Alexa Fluor 647 mAb (clone CC302, Bio-Rad Antibodies, Kidlington, UK) and TNF-α-Brilliant Violet 421 mAb (clone Mab11, BioLegend) as described previously [22 (link)]. Cells were analysed using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec).

Freshly isolated PBMC were seeded at 5 × 10⁵ cells/well in 96-well round bottom plates (Costar, from Thermo Fisher Scientific) and stimulated with 1 µg/mL NiV G or NiV F peptides pools in triplicate wells. Unstimulated cells in triplicate wells were used as a negative control. After 2 h at 37 °C, cytokine secretion was blocked using 1:1000 BD GolgiPlug (BD Biosciences) and cells were further incubated for 12 h at 37 °C, 5% CO₂. PBMC were then surface labelled with Zombie NIR fixable viability stain (Biolegend), CD3-FITC mAb (clone BB23-8E6-8C8, BD Biosciences), CD4-PerCP-Cy5.5 mAb (clone 74-12-4, BD Bioscience) and CD8α-PE mAb (clone 76-2-11, BD Bioscience). Following fixation (Fixation Buffer, BioLegend) and permeabilization (Permeabilization Wash Buffer, BioLegend), cells were stained with: IFN-γ-Alexa Fluor 647 mAb (clone CC302, Bio-Rad, Kidlington, UK) and TNF-α-Brilliant Violet 421 mAb (clone Mab11, BioLegend). Cells were analyzed using a MACSQuant Analyzer flow cytometer (Miltenyi Biotec). In defined experiments, cryopreserved PBMC from selected pigs were resuscitated, stimulated with peptides as described above, and surface stained with Zombie NIR, CD4-PerCP-Cy5.5 mAb, CD8β-PE mAb (clone PPT23, Bio-Rad), TCR1 delta chain mAb (clone PGBL22A, Kingfisher Biotech, Saint Paul, USA)/IgG1-Alexa Fluor 488 secondary Ab (BioLegend) prior to ICS.