Rbc lysing buffer

Polyfunctionality T cell responses were detected by intracellular cytokine staining using FC. At the end of survival, splenocytes were harvested and incubated with RBC Lysing Buffer (Solarbio, China) for 10 min at room temperature and then were washed and diluted with RPMI 1640 medium (Gibco-BRL) with 10% FBS into 96-well plates at 1 × 10⁶ per well. Peptides and brefeldin A (Sigma-Aldrich, USA) were added, respectively, at 1 nM and 1 μg/ml. After incubating for 16 hours at 37°C, splenocytes were surface-stained with anti-CD3 (EF506), anti-CD4 (EF450), and anti-CD8 (BV605) and intracellular-stained with IFN-γ (PE), TNF-α (FITC), and IL-2 (APC) after fixation/permeabilization. The stained samples were acquired through a CytoFLEX flow cytometer, and the data were analyzed by CytExpert software.

Poly-inosinic-polycytidylic acid (Poly IC) was purchased from Sigma-Aldrich (St. Louis, MO). CCK-8 cell proliferation and cytotoxicity assay kit, nicotinamide adenine dinucleotide (NAD), RBC Lysing Buffer, and other reagents were obtained from Solarbio. Tumor necrosis factor (TNF)-α, interferon (IFN)-β, interleukin (IL)-1β, IL-23, IL-17, and IL-22 ELISA kits were purchased from Neobioscience. Bradford Protein Assay Kit was obtained from Beyotime Biotechnology. For flow cytometry, Intracellular Fixation/Permeabilization Buffer Kit, purified anti-mouse CD16/32, FITC-CD11c, PE-MHC II, APC-CD80, PerCP-CD86, PE-F4/80, APC-CD11c, and PerCP-Ly6G were from Elabscience; FITC-lineage cocktail and APC-RORγt were from eBioscience. B5 (amino acid sequence: NPQSCRWNMGVCIPISCPGNMRQIGTCFGPRVPCCRRW) was synthesized at Shanghai Apeptide Co., Ltd. M.W.: 4325.12; Purity: >95%; B5 is soluble in water, grand average of hydropathy: −0.268; Isoelectric point: 8.91; B5 stock solution (2 mg/ml in PBS) for experiments. PBD2 (amino acid sequence: DHYICAKKGGTCNFSPCPLFNRIEGTCYSGKAKCCIR) was synthesized at Shanghai Apeptide Co., Ltd. Purity: >95%.