Foxp3 fixation permeabilization concentrate and diluent

Cells isolated from mLNs, colon LP and SI LP were divided into two groups for intracellular staining: (1) anti-IL17A + anti-RORγt; and (2) anti-FoxP3. Cells of the first group were stimulated for 4–5 h at 37 °C with PMA (50 ng/ml), ionomycin (750 ng/ml; both from Sigma-Aldrich), in the presence of 10 μg/ml of Brefeldin A (BD Biosciences, San Jose, CA, USA), to activate IL17A expression. After this period, all cells were washed with PBS solution and were stained for cell viability with Fixable Viability Dye eFluor® 780 (1:1000; eBiosciences, San Diego, CA, USA) for 30 min at RT. Cells were then stained with the following extracellular antibodies: anti-TCRb-PerCP-Cy5.5 (1:100; H57-597 clone; eBioscience) and anti-CD4 + -Pacific Blue (1:200, RM4-5 close, BD Biosciences) for 30 min at 4 °C. Cells were fixed and permeabilized with FoxP3 Fixation/Permeabilization Concentrate and Diluent (eBioscience) according to manufacturer’s instructions and were stained for 90 min at 4 °C, with the following intracellular antibodies: anti-IL17A-APC (1:100; TC11-18H10.1 clone, Biolegend, San Diego, CA, USA) and anti-RORγt-PE (1:100; AFKJS-9 clone; eBioscience); or anti-FoxP3-FITC (1:100; FJK-16s clone; eBioscience). The data was acquired on LSR II cytometer (BD Bioscience) and was analyzed with FlowJo software (10.2 version) (TreeStar, Ashland, OR, USA).

Splenocytes were pre-incubated with Fc-block (BD Pharmingen) and stained with anti- CD8α, CD4 and TCRβ antibodies. For immunodetection of HDAC1 and HDAC2, cells were fixed and permeabilized using Foxp3 Fixation/Permeabilization Concentrate and Diluent (eBioscience) according to the manufacturer’s instruction. Subsequently, cells were blocked in 5% normal goat serum and then incubated with rabbit anti-mouse HDAC1 and mouse anti-mouse HDAC2 antibodies in permeabilization buffer (eBioscience) for 1 hour. Cells were washed with permeabilization buffer and incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG1 and biotinylated anti-mouse IgG1 antibodies, followed by a Streptavidin secondary staining.

For flow cytometric analyses, 1.0x10⁶ maternal lymph node and uterus cells and 0.5x10⁶ PBMCs were used for immuno-phenotyping. Non-specific binding was blocked by rat anti-mouse CD16/CD32 Mouse Fragment crystallizable (Fc) Block (1:200, BD Bioscience) and Normal Rat Serum (1:100, eBioscience) for 15 min at 4°C. Subsequently, the cells were incubated with the respective antibodies for 30 min for surface and intracellular staining. Antibodies are summarized in Table 1. In order to identify dead cells, cells were simultaneously stained with eFluor 506 viability dye (eBioscience). For intracellular staining, cells were fixed and permeabilized using Foxp3 Fixation/Permeabilization Concentrate and Diluent (eBioscience) according to the manufacturer’s instructions.
In order to quantify the expression of the GR by flow cytometry, 2x10⁶ cells were first incubated with 10^-6 M progesterone or corticosterone or only medium for 15 minutes, respectively. Subsequently, samples were blocked and stained with the respective surface markers for 30 minutes. Afterwards, cells were fixed and permeabilized as stated above and intracellularly stained with an anti-GR antibody directly labelled with AF488 for 30 minutes.
Flow cytometric data were acquired using a BD LSRFortessa II (BD Biosciences) and analyzed using FlowJo (Tree Star, Ashland, OR, USA).