Anti foxp3 pe

Flow cytometry analysis was performed using a Cytek^® Aurora full spectrum flow cytometer (Cytek Biosciences, California, USA). Cryopreserved PBMCs were gently thawed with a mean recovery of 87.50% viable cells, assessed by the Zombie UV™ Fixable Viability Kit (BioLegend, BioLegend, San Diego, CA, USA). 0.5 × 10⁶ PBMCs were stained in duplicate by using anti-CD3 BV570 (UCHT1), anti-CD4 VioBright R720 (REA623), anti-CD25 PE-Vio615 (REA570), anti-CD45RA VioGreen (REA1047), anti-CD197 (CCR7) PE-Vio770 (REA108), anti-CD279 (PD-1) VioBright 515 (REA1165), and anti-FoxP3 PE (REA1253) (Miltenyi Biotec, Bergisch Gladbach, Germany). The Transcription Factor Staining Buffer Set (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for nuclear staining. Isotype controls were used to rule out unspecific binding of antibodies. Gating was performed using FlowJo^™ 10.8.1 (see Fig. 2 for gating strategy). T_regs were gated as CD4⁺CD25^highFoxP3⁺, PD-1⁺ effector T_regs (eT_regs) as CD4⁺CD25^highFoxP3⁺CD45RA⁻PD-1⁺, and naïve T_regs (nT_regs) as CD4⁺CD25^highFoxP3⁺CD45RA⁺CCR7⁺. Median fluorescence intensity (MFI) was calculated in FlowJo™ and used as a relative surrogate marker of protein level.Fig. 2

Gating strategy

To characterise circulating leukocytes by flow cytometry, 10 µL of heparinised whole blood was incubated for 30 min at RT with CD45-FITC (BD Biosciences, Franklin Lakes, NJ, USA) to identify leukocytes. Lymphocytes and neutrophils were identified by complexity and morphology, whereas monocytes were gated based on the CD115-APC marker (Biolegend, San Diego, CA, USA). Ly6C-PerCP (BD Biosciences, San Diego, CA, USA) and CD115-APC (Biolegend, San Diego, CA, USA) were used for Ly6C^low and Ly6C^hi-monocyte subsets identification. For circulating lymphocytes analysis, 10 µL of heparinised whole blood was incubated with 5 µL Brilliant Stain Buffer (BD Biosciences, San Diego, CA, USA) followed by CD4-BV421, CD8a-BV510, CD3e-APC, or CD69-PE antibodies (BD Biosciences, San Diego, CA, USA). Mouse Tregs were identified using Kit FoxP3 Staining Buffer Set and with anti-CD25-APC, anti-Foxp3-PE, and anti-CD4-VioBlue (all from Miltenyi, Bergisch Gladbach, Germany). The samples were incubated with FACS Lysing solution (BD Biosciences) for 10 min before flow cytometry analysis (FACSVerse or FACS Fortessa Flow cytometer, BD Biosciences, San Diego, CA, USA).

Cells were treated with FcR‐block (Miltenyi Biotec) and stained for cell surface markers. Cells were then treated with fixation/permeabilization buffer (eBioscience) to label intracellular transcription factors. T_REGcells: anti‐CD3‐APC‐Vio770 (Miltenyi Biotec; clone REA641), anti‐CD4‐FITC (Miltenyi Biotec; clone REA604), anti‐CD25‐PE‐Vio770 (Miltenyi Biotec; clone 7D4), and anti‐FoxP3‐PE (Miltenyi Biotec; clone REA788). T_H1 cells: anti‐CD3‐PE‐Vio770 (Miltenyi Biotec; clone REA641), anti‐CD4‐FITC (Miltenyi Biotec; clone REA604), anti‐CD183‐PE (Miltenyi Biotec; clone CXCR3‐173), anti‐T‐bet‐APC (Miltenyi Biotec; clone REA102). T_H17 cells: anti‐CD3‐APC‐Vio770 (Miltenyi Biotec; clone REA641), anti‐CD4‐FITC (Miltenyi Biotec; clone REA604), anti‐RORγT‐APC (Miltenyi Biotec; clone REA278), anti‐AHR‐PE‐Vio770 (eBioscience; clone 4MEJJ). Dead cells were excluded from analysis via e450 viability dye (Invitrogen). A minimum of 10,000 gated cells were analyzed per specimen. Data were acquired by the MACSQuant System (Miltenyi Biotec) and analyzed by FlowJo 11.0 software (TreeStar, Ashland, OR, USA).