Pecf594 cd4

A total of 2×10⁶ murine splenocytes were incubated in a 96 U bottom well (Sarstedt) along with brefeldin A (1:1,000) and monensin A (1:1,000) at 37 °C for 5 hours. Stimulants were added at 2 µg and 40 MOI of peptides or adenovirus, respectively. After stimulation, samples were stored at 4°C overnight. Samples were then processed by centrifuging and resuspending pellets in murine Fc-block (BioLegend) at 4°C for 15 min. Extracellular proteins were then stained using the following panel: BV711 CD3 (BD Biosciences, #563123), BV510 CD8 (BD Biosciences, #563068), PE-CF594 CD4 (BD Biosciences, #562285), APC CD62L (eBioscience, #17-0621-81), AF700 CD44 (BioLegend, #103026) and v450 CCR7 (BD Biosciences, #560805). After staining, samples were fixed and permeabilized using a Fixation/Permeabilization kit (BD Biosciences) and following the manufacturer’s instructions. Intracellular proteins were then stained with the following panel: BV650 TNF-α (BioLegend, #506333), PE-Cy7 IL-2 (BioLegend, #503832), PE IFN-γ (BioLegend, #505808) Perp-eFluor-710 (Invitrogen, #46-1541-82) and FITC IL-6 (BioLegend, #503806). Samples were then run on a Fortessa flow cytometer (BD Bioscience) and data was analyzed with FlowJo (FlowJo V.10.8.2)