Daan Pieren

32 Chapter 2 time kinetics in cytokine secretion assays as well as differences in strength of stimulation. In this study, we aimed to reveal the impact of ageing on T-cell responsiveness by assessing the in vitro response kinetics of cytokine secretion, activation marker upregulation, and proliferation of T cells of young and aged mice in response to antigen-independent stimulation. We found that despite low proliferative capacity, T cell subsets of aged mice do respond to stimulation by upregulation of activation markers and secretion of cytokines. Furthermore, dimensionality reduction (viSNE) [38] analyses allowed us to assess the phenotypical changes occurring in T cells over time and revealed increased variation in the responsiveness of T-cell subsets of aged mice. Our findings stress the importance of addressing T-cell response kinetics and the strength of stimuli used to characterize the impact of ageing on the T-cell compartment. MATERIALS AND METHODS Mice Young (2 months of age) and aged (age range groups: 17-18 months, 22-24 months, and 28 months of age) C57BL/6 mice were purchased from Envigo (Venray, Limburg, The Netherlands). Mice were maintained at the animal facilities of the Institute for Translational Vaccination (Bilthoven, Utrecht, The Netherlands). Ethics Animal studies were approved by the Animal Ethical Committee of the National Institute for Public Health and the Environment (DEC no. 201400042). All procedures were carried out in accordance with Dutch national legislation. Preparation of single cell suspensions and proliferation labelling Spleen single cell suspensions were prepared by homogenizing the spleen through a cell strainer. Red blood cells were lysed with ACK lysis buffer (0.155 M NH4Cl; 10 mM KHCO3; 0.1 mM Na2EDTA, pH 7.2-7.4). Labelling to track proliferation was performed as follows: splenocytes were resuspended in PBS to 10*10 6 cells/mL and then labelled with 0.5 μ M CellTrace TM Violet (Invitrogen, Carlsbad, CA, USA) in PBS per milliliter of splenocyte suspension for 20 minutes at 37⁰C. Cells were then washed twice with ice-cold RPMI (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) medium containing 10% fetal calf serum (FCS, Greiner Bio-One, Kremsmünster, Austria).