Anti cd3e fitc

BTK inhibition with PF-06250112 was evaluated on splenocytes labeled with anti-CD86-FITC (BD Pharmingen, San Jose, CA, USA), and anti-B220-PE (BioLegend, San Diego, CA, USA). Phenotypic analyses were performed using anti-CD4-Alexa 700, anti-CD19-Pacific blue (Biolegend), anti-CD45-APC (eBiosciences, San Diego, CA, USA); anti-CD3e-FITC, anti-CD11b-PE (BD Pharmingen), anti-CD21-APC Cy7, anti-CD23-PE Cy7 and anti-GL7 Alexa 488 (Biolegend). Fluorescence activated cell sorting analysis was done on live cells using BD LSRII and FACSDiva software.

The spleens of mice were placed in cold RPMI-1640 medium immediately and then cut into small pieces with eye scissors. Single-cell suspensions of spleen tissues were acquired after filtration with 300- and 100-mesh filters. Single-cell suspensions of bone marrow tissues were obtained from the thigh bones of mice after being washed with cold PBS. The red blood cells were then disrupted within the cell suspension. For treatment, 2×10⁶ cells were stimulated with phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO, USA; 50 ng/ml) plus 2 µg/ml ionomycin (Sigma) and monensin (Sigma; 5 µg/ml) for 5 h. The cells were then collected for Th17 cytokine staining. Subsequently, they were fixed and/or permeabilized using fixation/permeabilization kits (BD, USA) according to the manufacturer's instructions, and incubated with anti-interleukin (IL)-17A-Alexa Fluor (BD), anti-CD4-PE (BD), anti-CD4-APC (BD), anti-CD25-PE-CyTM7 (BD), anti-FoxP3-PE (eBioscience, San Diego, CA, USA), anti-CD3e-FITC (BD), anti-CD4-PE-CyTM⁷ (BD), anti-CD8-PE (BD), anti-CD11b-APC (BD), and anti-Gr-1-PE (BD) antibodies for 40 min at 4°C. The cells were incubated with rat anti-mouse CD16/CD32 (BD) for 30 min before CD11b and Gr-1 staining at 4°C. Flow cytometry (BD) was performed, and data were analyzed using FlowJo software.