Zombie nir live dead cell kit

The phenotypes of different T‐cell subsets derived from MLC experiments were assessed after 6 days of co‐culturing by FACS analysis using a set of cell surface markers and intracellular cytokines to characterize CD4⁺ T helper (Th) cells Th1 39, 40, Th2 39, 40, Th17 39, 40, Treg 41 and CD8⁺ cytotoxic T lymphocytes (CTL) 42, as reported in Table 2. Before fixation, samples were stained with Zombie NIR Live/Dead Cell Kit to remove dead cells from the analysis (eBiosciences, San Diego, CA, USA). Cells were then fixed, stained for surface antigens, permeabilized and stained for intracellular antigens as reported in 30. In parallel, cells from MLC experiments were also evaluated for T‐cell intracellular cytokine production. Therefore, cells were stimulated for 5 hrs at 37°C with phorbol‐12‐myristate‐13‐acetate (50 ng/ml), calcium‐ionomycin (CaI, 1 μg/ml) and Brefeldin A (10 μg/ml) (all from Sigma‐Aldrich), and viable cells were analysed on a flow cytometer.

T cells were cultured with macrophages as described above. After 6 days of co‐culture, T cell phenotype was investigated by flow cytometry. Samples were stained with Zombie NIR Live/Dead Cell Kit (eBiosciences, San Diego, CA, USA) and then fixed with 0.5% methanol‐free formaldehyde (ThermoFisher, Waltham, MA, USA) for 15 min at room temperature (RT). The surface staining was carried out for 30 min at RT, using the following anti‐human antibodies: CD4 BV421 (1:50, RPA‐T4), CD45RA FITC (1:80, HI100) or CD45RA PerCP‐Cy5.5 (1:80, HI100), CD25 PerCP‐Cy 5.5 (1:40, M‐A251) or CD25 BV421 (1:100, M‐A251), CD119 PE (1:80, GIR‐208), CD183 PE‐Cy7 (1:40, 1C6/CXCR3). Cells were then permeabilized with 0.05% Saponin/100 mm Tris–HCl, pH 7.4, for 15 min. The staining of intracellular antigens was performed by incubating cells for 30 min with anti‐human antibodies FoxP3 PE‐CF594 (1:30, 259D/C7) or GATA3 AlexaFluor 647 (1:20, L50–823; both from BD Biosciences). Cells were acquired with a FACSAria (BD Biosciences) and analysed using FCS express v. 4.07 (DeNovo Software). Gating strategies are shown in Figures S2B (Th1 and Th2) and S2C (Treg) (see supporting information).