Anti foxp3 apc

To analyze the potential effects of CPM on circulating Tregs, PBMCs were co-stained with anti-CD4-FITC (RPA-T4, BD Biosciences, San Jose, CA, USA) and anti-CD25-PE (BC96, eBioscience, San Diego, CA, USA). Then, the FoxP3/Transcription factor staining buffer set (eBioscience) was used to assess intracellular expression of FoxP3 (anti-FoxP3-APC) according to manufacturer’s instructions. Tregs were characterized as CD4+CD25^hiFoxP3+ lymphocytes, and were quantified as a percentage of circulating CD4+ T lymphocytes. Treg numbers in peripheral blood were also calculated by multiplying Treg frequencies in the lymphocyte population by absolute lymphocyte counts.

For flow cytometry, cells were washed in PBS supplemented with 1% FCS, 1% Sodium Azide and 3 mM EDTA (FACS buffer) and stained with a cocktail of anti-CD3 PE, anti-CD4 APC-Cy7 and anti-CD25 FITC (eBioscience). Cells were then fixed and permeabilized using eBioscience FoxP3 staining kit (77-5775-40) and stained with anti-Foxp3 APC. Samples were examined using a BD LSRFortessa flow cytometer and analyzed using FlowJo software (Treestar). For ELISA, supernatants from cell culture were removed and examined for their cytokine content using ELISA kits for IL10 (DY417) and IFNγ (DY485) according to the manufacturer's instructions (R&D systems). For Western blotting, cerebellar slices were scraped from the culture membrane and suspended in PTxE buffer (PBS, 1% Triton-x, 1 mM EDTA) using mechanical homogenisation and sonication. Western blotting was performed on tissue suspensions as we have described previously [30] (link). Primary antibodies used were: anti-MOG (Millipore, MAB5680), anti-myelin basic protein (MBP) (Abcam ab40390), anti-actin (ECM Biosciences, AM2021), anti-neurofilament H (NFH) (1/1000 dilution: Millipore, MAB5539). Secondary antibodies used were: HRP conjugated mouse (Sigma, A8924), and HRP conjugated rabbit (GE Healthcare, NA934).

Femoral bone marrow, mesenteric and liver (celiac, portal) lymph nodes cells were isolated, washed, and counted. Cells were treated with FcR-block (Miltenyi Biotec), surface stains were performed and intracellular stains were carried out following the fixation-permeabilization buffer manufacturer’s protocol (eBioscience). T_REGcells: anti-CD4-FITC (Miltenyi Biotec, clone GK1.5), anti-CD25-PE (eBioscience, clone PC61.5), anti-FOXP3-APC (eBioscience, clone FJK-16s). T_H17 cells: anti-CD4-FITC (Miltenyi Biotec, clone GK1.5), anti-CD196-PE (Miltenyi Biotec, clone REA277), anti- RORγt-APC (Miltenyi Biotec, clone REA278). T_H1 cells: anti-CD4-FITC (Miltenyi Biotec, clone GK1.5), anti-CD183-PE (Miltenyi Biotec, clone CXCR3-173), anti-T-bet-APC (Miltenyi Biotec, clone REA102). Dead cells were excluded via e450 viability dye (eBioscience). Data was acquired by the MACSQuant System. Analyses were performed via FlowJo VX software.

Tumor cells isolated from mice were thawed and stained for identification of myeloid or lymphoid cell subpopulations according to the procedure described by Rossowska et al. (30 (link)). Briefly, tumor-derived cells were stained with LIVE/DEAD Fixable Violet Dead Staining Kit (Thermo Fisher) and then stained with cocktails of fluorochrome-conjugated monoclonal antibodies: anti-CD3 PE-CF594, anti-CD19 PE-CF594, anti-CD49b PE-CF594 (all from BD Biosciences), anti-CD45 BV605, anti-CD11b PerCP-Cy5.5, anti-CD11c BV650, anti-F4/80 AlexaFluor 700, anti-Ly6C PE, anti-Ly6G APC-Cy7, anti-MHC II FITC, anti-CD86 PE-Cy7 (all from BioLegend) for myeloid cell identification, and anti-CD45 BV605, anti-CD3 BV650, anti-CD4 FITC, anti-CD8 APC/Fire 750, anti-CD25 PE, anti-CD44 PE-Cy7, anti-CD62L PerCP-Cy5.5 (all from BioLegend) for lymphocytes identification. Then, the cells were fixed using the FoxP3 Fixation Permeabilization Staining Kit (eBioscience). Tumor cells stained with myeloid or lymphocyte cocktail were additionally incubated with anti-CD206 APC (BioLegend) or anti-FoxP3 APC (eBioscience) antibodies, respectively. The analysis was performed using a FACS Fortessa flow cytometer with Diva software (Becton Dickinson).

Lymph node and spleen cells from mice were prepared after treatments with PSORI-CM02. Cells were stained for surface markers with anti-CD4-PE/CD25-FITC Abs and then an intracellular marker with anti-Foxp3-APC using intracellular fixation/permeabilization kit (eBioscience, San Diego, CA, USA). CD4+ Foxp3 Tregs were analyzed using a FACSCalibur (BD, Biosciences). Since we found that only ~94% of CD4+ CD25+ T cells were FoxP3-positive (Figure S1 in Supplementary Material) in imiquimod-induced psoriatic mice, we quantified CD4+ FoxP3+ rather than CD4+ CD25+ cell population and defined the former as Tregs.

Lymph node and spleen cells from mice were harvested after treatments. Cells were isolated and stained using surface markers (anti-CD4-PE, eBioscience) and then an intracellular marker (anti-Foxp3-APC, eBioscience) using intracellular fixation/permeabilization kit (eBioscience, San Diego, CA). And CD4+Foxp3 Tregs were analyzed using a FACSCalibur (BD, Biosciences).