Tnfα pe mp6 xt22

Seven days after immunization, single cell suspensions generated from spleens were subjected to ACK red blood cell lysis and counted using a Vi-Cell automated cell counter (Beckman Coulter). For in vitro stimulation assays, 1 × 10⁶ cells were incubated with 1 μg/ml peptide and 3 μg/ml brefeldin A for 5 h at 37°C in complete media (RPMI 1640 containing 10% FBS, 10 mM HEPES, 0.1 mM β-ME, 0.1 mM non-essential amino acids, 0.1 mM sodium pyruvate, 2 mM L-glutamine and penicillin-streptomycin). After stimulation, cells were surfaced-stained with CD8α-BV421 (clone 53.67, BioLegend), CD4-FITC (GK1.5, BioLegend), B220-PE-Cy7 (clone RA3-6B2, Tonbo), and a fixable viability dye (Ghost Dye Red 780, Tonbo) for 10 min at room temperature. After staining for surface antigens, cells were fixed and permeabilized with FoxP3 fixation/permeabilization buffers (Tonbo) for 15 min at room temperature. After fixation and permeabilization, cells were washed in perm/wash buffer and stained for intracellular cytokines using IFNγ-APC (XMG1.2, Tonbo) and TNFα-PE (MP6-XT22, BD Biosciences) diluted in perm/wash buffer for 30 min at room temperature. After a final wash, flow cytometry data were acquired on a four-laser (405, 488, 561, 638 nm) CytoFLEX S flow cytometer (Beckman Coulter) and analysis was performed using FlowJo (version 10.7.1; BD Biosciences).