Daan Pieren

34 Chapter 2 Dimensionality reduced analyses Dimensionality reduced analyses (viSNE) of flow cytometry data were performed in Cytobank (www.cytobank.com) [38]. Cell density maps of dimensionally reduced single-cell viSNE data showing clustering of CD4 + and CD4 - naive and memory populations or Th, Tc, and Treg-cell subsets of six pooled young mice (2 months old) and four pooled aged mice (28 months old) before and after receiving an intermediate strength of stimulation for four days. Expression of the designated cellular markers in the heatmaps was based on their ArcSinh5-transformed median expression. Indicated cluster frequencies were based on the cells present within the gates of the viSNE plots. The total viSNE plot of each T cell subset for each indicated day comprised 100% of cells of that subset. Cytokine assays in supernatant Supernatants of cell cultures were stored at -80⁰C and thawed before measuring the cytokines present. Secreted cytokines in culture supernatants were measured by using a Milliplex MAP kit Mouse Cytokine/Chemokine Magnetic Bead Panel (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. The following cytokines were measured: IL-2, IL-4, IL-5, IL-10, IL-17, IFN- γ , and TNF- α . Cytokines were detected on a Luminex (Bio-Rad, Hercules, CA, USA). Intracellular cytokine analysis For intracellular cytokine labelling, splenocyte suspensions (2*10 5 cells/well) of young and aged mice were cultured with phorbol 12-myristate 13-acetate (PMA) (0.05 μ g/mL) and ionomycin (0.5 μ g/mL) (Sigma Aldrich) in medium for four hours in total. After one hour of culturing, GolgiPlug (containing Brefeldin A; 1:1000, BD Biosciences) was added to allow intracellular accumulation of produced cytokines. After labelling with anti-CD4, anti-CD8, and Live/Dead Fixable Aqua, cells were fixed and permeabilized and labelled intracellularly with anti-CD3 and for the presence of IFN- γ , TNF- α , IL-4, and IL-5 as described in our flow cytometric labelling protocol. Statistical analyses Statistical analyses were performed using GraphPad Prism 7 software (La Jolla, CA, USA). Statistical significance was determined by using Mann-Whitney U test, Kruskal-Wallis test, or Two-way ANOVA. For all analyses, p values < 0.05 were considered statistically significant.