Kit foxp3 staining buffer set

The expression of T-bet and Eomes transcription factors was analyzed using cryopreserved PBMCs, thawed as indicated above. Surface staining was performed using anti-CD7 APC (clone: M-T701, BD Pharmingen), anti-CD56 BV421 (clone: NCAM16.2, BD Horizon), anti-CD16 PE- Vio770 (clone: VEP13, Miltenyi Biotec), anti-CD57 Biotin-Anti-Biotin-Viogreen (Miltenyi Biotec), and anti-CD3/anti-CD14/anti-CD19 conjugated with APC-Vio770 (clones: BW264/56, TÜK4, LT19 Miltenyi Biotec). After cell fixation and permeabilization using the Kit FoxP3 Staining Buffer Set (Miltenyi Biotec), following the manufacturer’s instructions, intracellular staining was realized with anti-T-bet PerCP Cy5.5 (clone: 04-46, BD Pharmingen) and anti-Eomes FITC (clone: WD1928, eBioscience) antibodies. Cells were then acquired on a 10 parameter MACSQuant cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany).
Data were analyzed using FlowJo v10 (Tree Star, Inc.). Once selected the CD7⁺CD3⁻CD19⁻CD14⁻ cells from PBLs singlets, three populations of NK cells were described: CD56^brightCD57⁻, CD56^dimCD57⁻, and CD56^dimCD57⁺. Then, the intracellular expression of T-bet and Eomes was measured in each of these three subpopulations (Figure S2 in Supplementary Material). FMO controls and flow cytometry compensation were performed as indicated above.

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).