Percp cy5.5 conjugated anti mouse cd4 antibody

The splenic cells were gently dispersed through nylon mesh into a single-cell suspension, washed with RPMI 1640 (Gibco, USA). Single cell suspensions were prepared by lysing red blood cells with red blood cell lysing buffer (BD Biosciences, Vienna, Austria). The splenic mononuclear cells were then resuspended in RPMI 1640 medium with 10% FCS (Gibco, USA), stimulated with phorbol myristate acetate (PMA, 25 ng/ml, Sigma-Aldrich Poole, UK) and ionomycin (1 μg/ml, Sigma-Aldrich) in the presence of GolgiPlug (1 μl/10⁶cells, BD Biosciences) on a 24-well culture plate at 37 °C. After 5 h incubation, the cells were harvested and stained with PerCP-Cy5.5 conjugated anti-mouse CD4 antibody (BD Biosciences). Then cells were stained intracellularly with anti-IL-9-PE (eBioscience, San Diego, CA, USA), anti-IL-17-PE (eBioscience, USA), PE-conjugated anti-IL22 mouse antibody (eBioscience, USA) and anti-IFN-γ-Alexa-Fluor®488 (BD Biosciences) mouse antibody after fixation and permeabilization (BD Biosciences).

Spleens were harvested on the 33rd day and a single-cell suspension of splenocytes was generated by passing 50-mesh stainless mesh. For OVA-specific cell proliferation and cytokine expression, the cells (2 × 10⁶ cells/mL) were treated with OVA (100 μg/mL) in complete RPMI-1640 medium (Thermo Fisher Scientific, Inc.) and incubated for 72 h. The cells were collected and centrifuged at 1000× g for 15 min at 4 °C. The supernatant was collected for the detection of specific cytokine expression. The levels of IL-10, IL-4, and IFN-γ were evaluated by ELISA kits (Thermo Fisher Scientific, Inc.). For intracellular cytokine detection, splenocytes were incubated with OVA (50 μg/mL) for 72 h. Brefeldin A (BFA) (BD Biosciences, San Jose, CA, USA) was added for 4 h before harvesting, and then the cells were washed twice with PBS and stained with a PerCP-Cy5.5-conjugated anti-mouse CD4 antibody (BD Biosciences). Cells were fixed with fixation buffer (Biolegend, San Diego, CA, USA) and permeabilized with wash buffer (Biolegend) before staining with PE-conjugated specific antibodies against murine IFN-γ, IL-4, and Foxp3 (BioLegend). Subsequently, all samples were analyzed by a BD Accuri™ 5 flow cytometer (BD Biosciences). The mean fluorescence intensity (MFI) from each sample was calculated using C6 Accuri system software (version 1.0.264.15, BD Biosciences).