Automacs magnetic bead cell separation technology

Naïve CD4⁺ T cells (CD4⁺ L-selectin^hi cells) were purified using AutoMacs Magnetic Bead cell separation technology (Miltenyi Biotech) from total lymph node cells isolated from unprimed mice with purity ranging from 98–99.9%. For in vitro activation, 5×10⁵ naïve CD4⁺ T cells were activated in the presence of plate-bound anti-CD3 (1 µg/ml) plus Th1- (200 U/ml IL-2, 40 U/ml IL-12, 10 µg/ml anti-IL-4) or Th17-(10 ng/ml TGF-β1, 50 ng/ml IL-6, 1 µg/ml anti-IFN-γ, 1 µg/ml anti-IL-4, 1 µg/ml anti-IL-2) promoting conditions. On day four the cultured T cells were collected and the percentage of viable cytokine positive cells assessed via flow cytometry. The cells were stained with LIVE/DEAD® Fixable Violet Dead Cell Stain Kit, for 405 nm excitation (Life Technologies), anti-CD4-APC/Cy7 (clone RM4–5), anti-IFN-γ-PerCP/Cy5.5 (clone XMG1.2), and anti-IL-17-APC (clone eBio17B7) (eBioscience). Viable cells (5×10⁵) were analyzed per individual sample using a BD Canto II cytometer (BD Biosciences), and the data were analyzed using FloJo Version 9.5.2 software (Tree Star, Inc).