Anti mouse ki67 b56

Naïve CD4⁺ T cells were incubated in T_reg cell differentiation condition in the presence of the following: plate-bound anti-CD3 (2 µg/ml, eBioscience), anti-CD28 (3 µg/ml, eBioscience), anti-IL-4 (10 µg/ml, BioXCell), anti-IFN-γ (10 µg/ml, BioXCell), anti-IL-12 (10 µg/ml, BioXcell), IL-2 100 U/ml, Peprotech) and TGF-β (5 ng/ml, R&D Systems). Cells were stained for surface markers with anti-mouse CD4 (RM4–5, eBioscience) and anti-mouse CD8 (53-6.7 eBiosciences). Intracellular staining was performed using Cytofix/Cytoperm buffer (eBioscience) to fix and permeabilize cells. Cells were then washed and stained with following antibodies: anti-mouse Helios (22F6, BioLegend), anti-mouse Ki67 (B56, BD Biosciences) and anti-mouse Foxp3 (FJK-16s, eBioscience). Dead cells were excluded from analysis using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Life Technologies). Sample were run on FACSCanto II cell analyzer (BD Biosciences) and analyzed with FlowJo software.