Fitc conjugated anti il 17 antibodies

For intracellular detection of IL-17, CD4⁺ T cells were incubated with 25 ng/mL phorbol 12-myristate 13-acetate (PMA), 250 ng/mL ionomycin (Sigma-Aldrich), and monensin-containing GolgiStop (BD Biosciences, San Jose, CA, USA) for 4 h. The harvested cells were stained with PerCP-conjugated anti-CD4 antibodies (Biolegend, San Diego, CA, USA). After fixation with fixation/permeabilization solution, the cells were stained with 0.125 μg FITC-conjugated anti-IL-17 antibodies (eBioscience, San Diego, CA, USA) to determine the population of Th17 cells. All analyses were performed using a BD LSRII fortessa (BD Biosciences) and FACS DIVA version 10.0 (BD Biosciences).

For immunostaining, 7-μm tissue sections of spleens were used. To analyze the populations of T helper 17 cells, we used PE-conjugated anti-CD4 and fluorescein isothiocyanate (FITC)-conjugated anti-IL-17 antibodies (eBioscience; San Diego, CA, USA). To analyze the populations of regulatory T cells, the samples were stained with FITC-conjugated anti-CD4, APC-conjugated anti-CD25, and PE-conjugated anti-Foxp3 antibodies. The stained sections were observed using a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) at × 400 magnification. Positive cells were counted, and the values were expressed as the mean ± standard deviation.

The cells used for analysis of the CD4⁺ IFNγ ⁺ (Th1), CD4⁺ IL-4⁺ (Th2), CD4⁺ IL-17⁺ (Th17), and IL-17-secreting CD19⁺ B cell (B17) populations were stimulated with PMA and ionomycin for 4 h using GolgiStop (BD Biosciences, Franklin Lake, NJ, USA). To quantify the Th1, Th2, Th17, and B17 cell populations, mouse splenocytes were immunostained using a PerCp5.5-conjugated anti-CD4 antibody (eBioscience, San Diego, CA, USA) for T cells and a PerCP5.5-conjugated anti-CD19 antibody for B cells. The cells were fixed and permeabilized using the Cytofix/Cytoperm Plus kit (BD Biosciences) following the manufacturer’s instructions, and then stained with APC-conjugated anti-IFNγ, PE-conjugated anti-IL-4, APC-conjugated anti-IL-10, and FITC-conjugated anti-IL-17 antibodies (eBioscience), respectively. All samples were analyzed using the Attune NxT Flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA). The flow cytometry data were analyzed using FlowJo™ software (FlowJo, Ashland, OR, USA).