Anti cd127 apc

Cells were incubated with an anti-CD16/CD32 monoclonal antibody (mAb, eBioscience, 14-0161-82, 93, 1:33) and then stained with the following dye or anti-mouse antibodies: viability dye APC-Cy7 (eBioscience, 65-0865-14), anti-lineage cocktail-FITC (BioLegend, 133302, 145-2C11; RB6-8C5; RA3-6B2; Ter-119; M1/70, 1:40), anti-TCRβ-FITC (BioLegend, 109206, H57-597, 1:200), anti-CD90.2-PE/Cy7 (BioLegend, 105326, 30-H12, 1:200), anti-CD127-APC (BioLegend, 135012, A7R34, 1:100), anti-ST2-BV421 (BioLegend, 145309, DIH9, 1:100), anti-CD3E-FITC (BioLegend, 100203, 17A2, 1:200), anti-TCRg-PE (BioLegend, 118107, GL3, 1:100). Gating was performed as follows: ILC2s were defined as live Lin⁻ (CD3e⁻Ly6G⁻Ly6c⁻CD11b⁻CD45R⁻TER119⁻TCRb⁻) CD127⁺Thy1⁺ST2⁺ cells. Stained cells were examined with a FACSCanto II flow cytometer (BD Biosciences), and the results were analyzed using FlowJo software version 10.4.2 (TreeStar, FlowJo).

Specific antibodies against CD3-PE-Cy7 (Biolegend) or anti-CD3-FITC (Biolegend), anti-CD25-PE (BD Pharmingen™), anti-CD127-APC (Biolegend), anti-CD4-FITC (Biolegend), anti-CD8-APC (Biolegend), anti-CD45RA-FITC (Biolegend), anti-CCR7-PE-Cy7 (Biolegend), anti-CD4-PE (BD Biosciences), anti-PD-1-FITC (Biolegend), anti-CD4-PErCP-Cy5.5 (BD Biosciences), anti-Tim-3-PE (BD Pharmingen™), anti-FoxP3-PE (Biolegend), and anti-IFN-γ-APC (eBioscience) were used. 7AAD viability staining (Biolegend) was used to assess the viability of the cells. Tils were examined at day 0, 5, 10, 15, 20 and 25 by flow cytometry. Briefly, 1 × 10⁶ T cells was mixed with 5 μl of each antibody and was incubated on ice for 20 min in the dark. After incubation the samples were washed with FACS buffer (5% BSA in PBS, 0.09% sodium azide). The pellets were suspended in 300 μl of FACS buffer and acquired on a BD FACS Conto II flow cytometer, and then the data was analyzed with FlowJo software (TreeStar Inc). The data is displayed as background-corrected values with control sample or by fluorescence minus one (FMO).