Fixation permeabilization buffer kit

Hepatic and adipose immune cells were pre‐incubated with anti‐mouse CD16/32 Fc blocker (BD Pharmingen, USA), followed by staining with the Live/Dead marker anti‐FVD‐APC‐Cy7 (all supplied by eBioscience, San Diego, CA, USA). The fluorochrome‐conjugated antibodies used in this study were anti‐CD45, anti‐CD3, anti‐NK1.1, anti‐CD4, anti‐CD8, anti‐CD44, anti‐CD62L, anti‐CD11b, anti‐F4/80, anti‐Ly6C, anti‐Ly6C, and anti‐Siglec‐F (all supplied by eBioscience, San Diego, CA, USA). Liver mononuclear cells were stimulated with phorbol‐myristate acetate/ionomycin/brefeldin A/monensin for 5 hr in vitro. The cells were fixed and permeabilized using a Fixation/Permeabilization Buffer kit (eBioscience, San Diego, CA, USA). The permeabilized cells were washed with FACS buffer and resuspended in 1% formaldehyde and stained for intracellular cytokines with anti‐IFN‐γ‐PE‐Cy7, anti‐TNF‐α‐APC, and anti‐IL‐17A‐APC fluorochrome‐conjugated antibodies. Stained cells were analyzed using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA), and data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR, USA).

Cells were incubated with saturating concentrations of fluorochrome-conjugated mAbs for 30 min at 4 °C in the dark, washed, and collected for FCA. In the case of biotin-conjugated/unconjugated Abs, the cells were incubated for a further 30 min with appropriate reagents for the second step. To determine cell apoptosis, after labeling of the surface markers, SC cells were washed with PBS and then with Annexin V binding buffer (BD Biosciences Pharmingen). After washing, the cells were incubated with 5 μL of Annexin V-FITC (BD Biosciences Pharmingen) for 15 min at room temperature in the dark and collected for FCA. For IL-17 and IFN-γ staining, cells were restimulated with phorbol 12-myristate 13-acetate and 400 ng/mL ionomycin for 4 h in the presence of 3 μg/mL of brefeldin A. Afterwards, the cells were fixed and permeabilized overnight using the fixation/permeabilization buffer kit (eBioscience) and then stained with fluorochrome-conjugated mAbs. For sample acquisition, the FACSVerse flow cytometer (Becton Dickinson, Mountain View, CA, USA) and FlowJo software version 7.8. (TreeStar Inc., Ashland, OR, USA) were used for the analyses. Appropriate fluorescence minus one controls or isotype controls were used to establish gates for cell marker positivity.

Following stimulation with PMA and ionomycine, MACS-separated SC and dLN CD4+ T cells and surface-stained SC and cultivated dLN mononuclear cells were fixed/permeabilized overnight at 4°C, using the fixation/permeabilization buffer kit (eBioscience), according to the manufacturer’s instructions (eBioscience; http://www.ebioscience.com/resources/best-protocols/flow-cytometry-protocols.htm). For intracellular cytokine content assessment, washed cells were stained with fluorochrome-conjugated mAbs to IL-17, IFN-γ and GM-CSF for 30 min at room temperature in the dark, washed again and collected for FCA.
For detection of CD4+CD25+ Treg lymphocytes, dLN cells were surface-stained for CD4 and CD25, fixed/permeabilized using the reagents from the Foxp3 Staining Set (eBioscience), and stained with FITC-conjugated anti-FoxP3 mAb, as suggested by the manufacturer.

After surface antigen immunolabeling, thymocytes and SC cells were fixed/permeabilized using the reagents from the fixation/permeabilization buffer kit (eBioscience), and stained with anti-Foxp3, anti-Ki-67, anti-granzime B or p16^INK4a/anti-mouse IgG Abs, as suggested by the manufacturer.

Cells were washed with PBS and incubated with antibodies diluted in PBS/10% HS/0.01% NaN3 (sodium azide) for 30 min on ice. Surface antibodies were anti-CD4 PerCp (clone SK3), CD3 FITC (clone UCHT1), and CD25 PE (clone M-A251, all BD Biosciences). Intracellular staining with anti-Foxp3 APC (clone PCH101, eBiosciences) was performed using the eBioscience fixation/permeabilization buffer kit. A minimum of 10⁵ events in the lymphocyte gate was acquired using a FACScalibur flow cytometer for 4-color analysis and analyzed using WEASEL software (WEHI, Melbourne, VIC, Australia). Cells were gated first based on forward and side scatter to excluded dead cells and cell debris. T cells in the lymphocyte gate were identified based on CD3 expression, further sub-gated on CD4+ T cells (Figure 1A) and CD25+ cells then subdivided into Foxp3hi and Foxp3int cells (Figure 1B).

Spinal cord and lymph node mononuclear cells stimulated with PMA and ionomycin were stained for surface TCRαβ and CD8 antigens and then washed and fixed/permeabilized overnight at 4 °C, using the solutions from fixation/permeabilization buffer kit (eBioscience; http://www.ebioscience.com/resources/best-protocols/flow-cytometry-protocols.htm). Next, for double IFN-γ/IL-17 staining, the cells were washed using appropriate permeabilization buffer (eBioscience), incubated with fluorochrome-conjugated anti-IFN-γ and anti-IL-17 mAbs for 30 min at room temperature in the dark, and then washed again and collected for FCA. For GM-CSF analyses, the fixed MACS-sorted CD4+ cells were sequentially labeled using anti-GM-CSF mAb, PerCP/Cy5.5-conjugated goat anti-mouse IgG and fluorochrome-conjugated anti-IFN-γ/anti-IL-17 mAb cocktail, as described above.

To determine the frequency and phenotype of T cell populations in PBMCs, multicolor flow cytometry was performed using the following surface antibodies: anti-CD3 PerCP (BD Pharmingen, USA) and anti-CD8 FITC (Biolegend, USA); anti-CD4 APC-Cy7 (BD Pharmingen, USA), and anti-CD25 PECF584 (BD Pharmingen, USA), anti-CD127 BV650 (Biolegend, USA), anti-TNFR2 biotinylated followed by conjugation with Streptavidin PECy7 (BD Pharmingen, USA), anti-PD-L1 PE (Biolegend, USA), anti-CTLA-4 BV605 (Biolegend, USA), and anti-GARP BV711 (BD Pharmingen, USA). Following primary staining, a fixable dead cell marker (Life Technologies, USA) was also used to distinguish between dead and live cells. Intracellular levels of FoxP3 and IFN-γ were determined following fixation and permeabilization of cells using a fixation/permeabilization buffer kit (eBioscience, USA) then staining the cells with anti-FoxP3 APC (eBioscience, USA) and anti- IFN-γ v450 antibody (eBioscience, USA). Flow cytometry data were acquired on a Becton Dickinson LSR II flow cytometer using FACS Diva software, acquiring a minimum of 100,000 events per sample. Fluorescence minus one (FMO) controls and isotype-matched immunoglobulins were used to enable accurate gating. Data were analyzed using Flow jo software (TreeStar, USA).