Anti foxp3 fitc

Levels of cytokines and transcription factors were assessed by intracellular staining using anti-IL-17-FITC, anti-Foxp3-FITC, and anti-Foxp3-PE antibodies (all from eBioscience, San Diego, CA, USA). Cells were stimulated with phorbol myristate acetate and ionomycin with the addition of GolgiStop for 4 h. Cultured cells were surface labeled for 30 min and permeabilized with Cytofix/Cytoperm solution (BD Pharmingen, Heidelberg, Germany). Cells were intracellularly stained with fluorescent antibodies and subjected to flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Events were collected and analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Antibodies used for cell staining were pre-titrated and used at optimal concentrations. A FACS Aria flow cytometer (Becton Dickinson, USA) and FlowJo software was used for analysis. For FACS purification LCs were stained for CD207 (anti-CD207 PeVio700), CD1a (anti-CD1a VioBlue) and HLA-DR (anti-HLA-DR Viogreen, Miltenyi Biotech, UK). For T cell staining, antibodies anti-CD3 PerCP, anti-CD4 Viogreen, anti-CD127 Pe (Miltenyi Biotech, UK) and anti-CD25 PeCy7 (Invitrogen, UK) were used for surface staining. Anti-FOXP3 FITC (eBiosciences, UK), anti-IL-10 PE (Miltenyi, UK) and anti-IDO1 AlexaFluor647 (Biolegend, UK) antibodies were used for intranuclear and intracellular staining. IDO1 intracellular staining of LCs was performed using Intracellular Fixation & Permeabilization Buffer Set (eBioscience, UK), following manufacturer protocol.

Cells isolated from the BAL and other tissues were analyzed via flow cytometry using the following monoclonal antibodies: anti-CD8a-FITC (53-6.7), anti-CD25-PE (PC61), anti-CD4-PeCy7 (RM4-5), anti-CD3e-PerCP-Cy5.5. (145-2C11), and anti-CD19-PerCP-Cy5.5 (1D3) (BD PharMingen, San Diego, CA, USA). Samples were washed in PBS containing 0.2% bovine serum albumin and 0.1% NaN₃. Aliquots containing 10⁴–10⁶ cells were incubated with 100 μl of appropriately diluted antibodies for 30 min at 4°C. After staining, the cells were washed with the above PBS solution, and relative fluorescence intensities were determined on a four-decade log scale by flow cytometric analysis using a LSR II (Becton-Dickinson, San Diego, CA, USA). For the identification of T_reg, intracellular staining of Foxp3 protein was used. Briefly, cells stained with the antibodies mentioned previously (i.e., anti-CD3e, anti-CD4, and anti-CD25) were permeabilized using fixation/permeabilization buffer following the manufacturer’s protocol, and stained using either anti-Foxp3-FITC (FJK-16s) or anti-Foxp3-APC (FJK16s) with corresponding isotype controls, IgG2a-FITC or -APC (eBioscience, San Diego, CA, USA).

For the analysis of cell surface markers, single-cell suspensions from the spleens were prepared 1 week after the last dose of DMBA. Cells were permeabilized by 0.5% (w/v) saponin in PBS (containing 0.25% (w/v) bovine serum albumin and 0.02% (w/v) sodium azide). Splenocytes (macrophages, B cells, CD4⁺ T cells and CD8⁺ T cells) were analyzed by staining with an antibody solution containing anti-CD11b-Alexa Fluor 700, anti-B220-Pacific Blue, anti-CD4-Dye647 and anti-CD8-PE/Cy7 (all obtained from eBioscience, Frankfurt, Germany). Tregs were analyzed by staining with anti-CD4-APC/Cy7 and anti-CD25-Dye647 and subsequent incubation with FoxP3 Fixation/Permeabilization working solution (all obtained from eBioscience). Then cells were stained with anti-FoxP3-FITC (eBioscience). All samples were measured using the flow cytometer BD LSR II (BD Biosciences, San Jose, CA, USA).