Pe conjugated anti foxp3 antibody

LP and MLN cells were assessed by FACS for specific surface markers using PE-conjugated anti-CD11c (1:100, Biolegend, cod. 117308), FITC-conjugated anti-CD103 (1:100, Biolegend, cod. 121419), FITC-conjugated anti-CCR7 (1:100, Serotec, cod. MCA2367F), APC-conjugated anti-CD3 (1:100, Miltenyi, cod. 130-117-793) Pacific Blue-conjugated anti-CD4 (1:100, Biolegend, cod. 100428), and FITC-conjugated anti-CCR9 (1:100, Miltenyi, cod. 130-115-602) antibodies. Cells (5 × 10⁵) were resuspended in 100 μl PBS containing 0.5% BSA and stained with the antibodies for 20 min at room temperature. Intracellular staining on LP and MLN cells was performed using Cytofix/Cytoperm, Fixation/Permeabilization Solution Kit (Becton–Dickinson), according to the manufacturer’s protocol. Briefly, cells were incubated with FITC-conjugated anti-CD4 and APC-conjugated anti-CD25 (1:100, Biologend, cod. 101709) antibodies, fixed/permeabilized for 15 min at 4°C, and processed for intracellular staining using PE-conjugated anti-FoxP3 antibody (1:100, Biolegend, cod. 126403) for 30 min at 4°C. Data were acquired on a FACS Canto II (Becton–Dickinson) and analyzed using DIVA 6.1 software.

To detect CD4⁺Foxp3⁺ Treg cells in the spleen and MLN, 2 × 10⁷ of splenocytes and MLN cells were pretreated by True-Nuclear Transcription Factor Buffer Set (Biolegend, San Diego, CA, USA) and then incubated with allophycocyanin (APC)-conjugated CD4 antibody (Biolegend) and PE-conjugated anti-Foxp3 antibody (Biolegend).
For CD4⁺IL17⁺ Th17 cells in the spleen and MLN, 2 × 10⁷ of splenocytes and MLN cells were incubated with allophycocyanin (APC)-conjugated CD4 antibody (Biolegend) and phycoerythrin (PE)-conjugated anti-CD17 antibody (eBioscience, San Diego, CA, USA).
The fractions of Treg, and Th17 cells from each tissue were analyzed using a FACSCalibur Flow Cytometer using the CellQuest software (Becton Dickinson, San Jose, CA).