Pe conjugated anti foxp3

For immunostaining, 7 μm tissue sections of spleens were stained. To analyze the populations of T helper cells, we used Alexa 488 conjugated anti-CD4, PE-conjugated anti-IL-17, APC-conjugated anti-CD25, and PE-conjugated anti-Foxp3 antibodies (eBiosciences, San Diego, CA, USA). To analyze the populations of STAT, AMPK and mTOR, the samples were stained with Alexa 488 conjugated anti-CD4, PE-conjugated anti-phosphorylated STAT-3 tyrosine 705, PE-conjugated anti-phosphorylated STAT-3 tyrosine 727, anti-mTOR, anti-AMPK, and anti-FGF21 antibodies, and anti-rabbit IgG-PE secondary antibody. The nuclei were stained with 4′,6-diamidino-2-phenylindole. The stained sections were analyzed using a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) at × 400 magnification. Positive cells were counted, and the numbers expressed as the mean±s.d.

For analyses of the phenotype and frequency of each cell subset, peridin chlorophyll protein (PerCP)-conjugated anti-CD4, phycoerythrin (PE)-conjugated anti-CD45RO, fluorescein isothiocyanate (FITC)-conjugated anti-interferon (IFN)-γ, allophycocyanin (APC)-conjugated anti-IL-17a, PE-conjugated anti-IL-23R, FITC-conjugated anti-CD4, APC-conjugated anti-CD25, PE-conjugated anti-FoxP3, APC-conjugated anti-CD39 (all from eBioscience, American) and phycoerythrin cyanin-7 (PE-Cy7)-conjugated anti-CD45RA (BD Bioscience) were used. For intracellular IL-17a and IFN-γ staining, cells were stimulated with 50 ng/mL phorbol myristate acetate (PMA) and 1 μg/mL ionomycin (both from Sigma-Aldrich) in the presence of 10 μg/mL GolgiStop (BD Bioscience) for 5 hours at 37 °C in a humidified 5% CO₂ incubator before staining. The cells were extracellularly stained, then treated with Cytofix/Cytoperm solution and Perm/Wash solution (BD Bioscience). For intranuclear FoxP3 staining, cells were first extracellularly stained and then treated with Fixation/Permeabilization and Permeabilization Buffer (FoxP3 Staining Buffer Set, eBioscience, American). The appropriate isotype controls were used in all the staining procedures, and the stained cells were processed with a BD FACS Calibur and analysed using FlowJo software, version 7.6.1.

Cells were stained with Percp-conjugated anti-CD4 Ab (BD Pharmingen), then stained with APC-conjugated anti-CD25, PE conjugated anti-Foxp3 and FITC-conjugated anti IL-17 (all from eBiosciences, San Diego, CA, USA), followed by fixation and permeabilization using the Buffer Set (BD Biosciences) according to the manufacturer's instructions. All samples were run on a FACSCalibur (BD Pharmingen), and data were analyzed using the FlowJo software (Tree Star, Ashland, OR, USA).

Cells were resuspended in fluorescence-activated cell sorting (FACS) buffer (5% fetal calf serum plus 5 mM EDTA in PBS) and incubated with conjugated monoclonal antibodies (mAb). The mAb used, phycoerythrin (PE)-conjugated anti–Ly-6G (clone RB6-8C5, 125931, 1/400), fluorescein isothiocyanate (FITC)-conjugated anti-CD11b (clone M1/70, 11011241, and 1/200), and allophycocyanin (APC)-conjugated anti-F4/80 (clone BM8, 17480182, 1/800), eF450-conjugated anti-CD4 (clone RM4-5, 48004282, 1/200), PE-conjugated anti–Foxp3 (clone FJK‐16 s, 12577382, 1/100), FITC-conjugated anti-CD25 (clone PC61.5, 53025382, 1/400), APC-conjugated anti-Arginase-1 (clone A1exF5, 17369782, 1/100), APC-conjugated anti-EGR2 (clone erongr2, 17669182, 1/100), were all from eBioscience. PE-conjugated anti-CD206 (clone CD68C2, 141705, 1/100) and PE-conjugated anti-IL-6 (clone MP5-20F3, 504504, 1/100) were from Biolegend. Cells (1 × 10⁶) were incubated with appropriate conjugated antibodies for 30 min at 4 °C in the dark. Stained cells were subsequently washed twice with FACS buffer and fixed in BD CellFIX solution (BD Biosciences). Flow cytometric analyses were performed on LSRII cytometer (Becton Dickinson) using FACS Diva6 (Becton Dickinson) and FlowJoX (Tree Star) software for data processing.

Cell pellets were prepared from the spleens of spondyloarthritic SKG mice. To examine the population of T helper cells, the cells were stained with PerCP-conjugated anti-CD4 (catalog number 45-0042-82) and APC-conjugated anti-CD25 (catalog number 102012) antibodies (eBioscience; Biolegend, San Diego, CA, USA), and then permeabilized and fixed with CytoFix/CytoPerm (BD Biosciences, San Diego, CA, USA) according to the manufacturer’s instructions. The cells were then further stained with PE-conjugated anti-FoxP3 (catalog number 12-5773-82), APC-conjugated anti-interferon (IFN) (catalog number 505810), and FITC-conjugated anti–IL-17 (catalog number 11-7177-81) (eBioscience; Biolegend, San diego, CA, USA).

To analyze intracellular cytokines, splenocytes were stained with PerCP-conjugated anti-CD4, APC-conjugated anti-CD25, FITC-conjugated anti-IL-17, and PE-conjugated anti-Foxp3 (eBiosciences), followed by fixation and permeabilization with a Foxp3 staining buffer kit (BD Bioscience) according to the manufacturer's instructions. Four hours before the staining, the cells were stimulated with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) (all from Sigma-Aldrich) and GolgiStop (BD Bioscience). All data was analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Adult mouse colon was separated into 0.5–1 cm segments and embedded in O.C.T. embedding compound. 8 µm sections were taken using a cryostat, fixed using 4% paraformaldehyde and washed using distilled water and TBST. Blocking solution containing 0.5% Triton X-100 was added for 15 min followed by an incubation with FITC-conjugated anti-CD45 (1:100; eBioscience, San Diego, CA, USA) and PE-conjugated anti-FoxP3 (1:50; eBioscience, San Diego, CA, USA) antibodies for 48 h at 4°C. Samples were mounted with ProLong™ Diamond Antifade Mountant with DAPI (Life technologies) and left to cure for 2 h. Samples were imaged the same day using a Nikon C1 (Upright) confocal microscope equipped with a 40× objective lens and running NIS Elements Software (Nikon. Tokyo, Japan). Images were analyzed using ImageJ 2.0 software. CD45⁺FOXP3⁺ cells were considered as regulatory T cells (Tregs).