Cell activation cocktail with brefeldin a

After a 5-days Th17 differentiation, the cells were stimulated with the Cell Activation Cocktail with Brefeldin A (BioLegend, San Diego, CA, USA) for 6 hours. Following activation of cells with this cocktail, the cell culture supernatant was collected and concentrations of IL-17 and IL-1β were measured by ELISA as previously described. For intracellular detection of the secreted cytokines, the activated cells were stained with APC anti-mouse CD4 antibody (Clone GK1.5; BioLegend, San Diego, CA, USA) and then treated with Intracellular Fixation and Permeabilization Buffer Set and FITC anti-mouse IL-17A antibody (Clone TC11-18H10.1; BioLegend, San Diego, CA, USA). Thereafter, the cells were examined by the Accuri C6 flow cytometer.

Cells were incubated with antibodies 4 °C 20 min. Cells were examined by Aria II Flow Cytometer (BD Bioscience, USA). Cells were gated as follows: DCs (F4/80⁻ CD11c⁺ IA/IE⁺), macrophages (CD11b⁺ F4/80⁺) and M1 macrophages (CD11b⁺ F4/80⁺ CD206⁻ MHC II^high), M2 macrophages (CD11b⁺ F4/80⁺ CD206⁺ MHC II^low). Intracellular cytokine staining: 2 µL mL⁻¹ Cell Activation Cocktail (with Brefeldin A) (Cat: 423,303, Biolegend, USA) was used to incubate cells at 37 °C in a CO₂ incubator for 6 h. Then, the Fixation/Permeabilization Solution Kit (Cat: 554,714, BD Biosciences, USA) was used to stimulus cells. After cell fixation and permeabilization (fixation/permeabilization solution, 100 uL/10⁶ cells, 4 °C, 30 min), the BD Perm/Wash™ Buffer is used to wash the cells and to dilute the IFN-γ antibody for staining (4 °C, 30 min). Antibodies: The following were purchased from BioLegend: anti-IA/IE (1:3200, Clone: M5/114.15.2, Cat: 107,630), anti-CD206 (1:200, Clone: C068C2, Cat: 141,705), anti-Ki67 (1:200, Clone: 16A8, Cat: 652,403), anti-IFN-γ (1:100, Clone: XMG1.2, Cat: 505,805). The following were purchased from eBioscience: anti-CD11b (1:200, Clone: M1/70, Cat: 47–0112-82), anti-CD11c (1:200, Clone: N418, Cat: 45–0114-82), anti-F4/80 (1:200, Clone: BM8, Cat: 17–4801-82).

The human monoclonal antibodies (mAbs) used for flow cytometry in this article included FITC-anti-CD3, FITC-anti-CD80, FITC-anti-CD163, FITC-anti-CD8α, FITC-anti-CD14, PE-anti-CD40, APC-anti-IFN-γ (all from BioLegend), BB515-anti-TLR2, and PE-anti-CD206 (all from BD Biosciences). Cells were scrapped from culture plates and incubated with human AB serum to saturate non-specific mAb binding before staining with the indicated fluorophore-labeled mAbs. For intracellular IFN-γ staining, cells were stimulated with Cell Activation Cocktail (with Brefeldin A; BioLegend) for 6 h according to the manufacturer's protocol. All data were collected using Novocyte flow cytometer (ACEA) and analyzed with FlowJo software.

Splenocytes were incubated with TruStain FcX™ PLUS anti-mouse CD16/CD32 (Biolegend; clone: S17011E) for 10 min at 4°C, and then stained with mAbs specific for various cell surface markers. To evaluate the production of IFN-γ and Granzyme B, splenocytes were diluted to 4 × 10⁶ cells/mL and cultured (500 μL/well) in a 24-well plate containing Cell Activation Cocktail (with Brefeldin A; Biolegend) for 6 h. Cells were then harvested and washed twice in cell staining buffer prior to surface marker staining as described above. Cells were then fixed and permeabilized using Intracellular Fixation Buffer & Intracellular Staining Permeabilization Wash Buffer (Biolegend). Intracellular staining was then performed using mAbs specific for IFN-γ and granzyme B, respectively. For Ki-67 staining, freshly isolated splenocytes were stained following the above-mentioned surface and intracellular staining procedures, with no in vitro stimulation. Samples were resuspended in cell staining buffer, tested with BD FACSCelesta flow cytometer, and analyzed using FlowJo software (BD, USA).

Single‐cell suspensions were prepared from spleens, pLNs, or peritoneal lavage fluids for FACS assays via LSRFortessa (BD Biosciences) and further for statistical analysis via FlowJo software. Cells were stained with fluorochrome‐labeled antibodies following incubation with anti‐CD16/CD32 to block Fc receptors. For intracellular cytokine (IFN‐γ and IL‐17a) detection, cells were incubated with anti‐CD16/32 and surface staining 6 h after Cell Activation Cocktail (with Brefeldin A) (Biolegend) stimulation. Then, cells were subjected to fixation/permeabilization kit (BD Biosciences) for intracellular cytokines staining. FoxP3/Transcription Factor Staining Buffer Set (Thermo) was used for detection of FoxP3 expression according to the instruction.