Daan Pieren

33 Novel features of ageing in murine T cells In vitro antigen-independent stimulation and splenocyte culture Soluble anti-CD3 (clone 145-2C11, eBioscience, San Diego, CA, USA) and anti- CD28 (aCD28, clone PV-1, SouthernBiotech, Birmingham, AL, USA) were used to stimulate cells to a low (0.019 μ g/mL anti-CD3), intermediate (0.019 μ g/mL anti-CD3 + 0.5 μ g/mL anti-CD28), or high (0.5 μ g/mL anti-CD3 + 0.5 μ g/mL anti-CD28) extent over time. For proliferation assays in response to exogenous IL-2, 0.1 μ g/mL recombinant murine IL-2 (eBioscience) was added with and without the presence of low anti-CD3 (0.019 μ g/mL). Stimulatory conditions were prepared in RPMI medium containing 10% FCS and then added to the splenocyte suspensions (4*10 5 cells/well) before incubation in 96-well U-bottom plates (CELLSTAR, Greiner bio-One) at 37⁰C for up to four days. Immunofluorescence labelling and flow cytometric analyses Single cell suspensions were washed and labelled at 4⁰C for a combination of cell surface markers with the following antibodies: anti-CD4-BUV395 (clone GK1.5), anti-CD8a-V450 (clone 53-6.7), anti-CD122-PE-CF594 (clone TMbeta1), anti-CD44-V450 (clone IM7) and anti-CD69-BV786 (clone H1.2F3) (BD Horizon, Franklin Lakes, NJ, USA); anti-CD69-PerCP-Cy5.5 (clone H1.2F3) and anti-PD- 1-BV785 (clone 29F.1A12) (BioLegend, San Diego, CA, USA); anti-CD25-PECy7 (clone PC61.5) (eBioscience); and Live/Dead Fixable Aqua (Invitrogen). Cells were labelled intracellularly with the following antibodies according to the FoxP3 Transcription Factor staining buffer set protocol (eBioscience): anti- CD3zeta-FITC (clone H146-968) (Abcam, Cambridge, Cambridgeshire, UK); anti-CTLA-4-BV605 (clone UC10-4B9), and anti-TNF- α -BV785 (clone MP6- XT22) (BioLegend); anti-FoxP3-eFluor660 (clone 150D/E4), anti-GARP-PE (clone YGIC86), anti-IFN- γ -PE-Cy7 (clone XMG1.2), and IL-4-PE (clone 11B11) (eBioscience); anti-IL-5-PE (clone TRFK5) (BD Pharmingen). Labelled cells were detected on a BD LSRFortessa X-20 (BD Biosciences, Franklin Lakes, NJ, USA). Gating analyses were performed using FlowJo software (Tree Star, Ashland, OR, USA). Cell proliferation Proliferation of cells was measured using detection of CellTrace by flow cytometry. As reported earlier [39], the fold change proliferation was calculated by dividing the CellTrace median fluorescent intensity (MFI) of the medium control by the stimulated cells of each individual animal. 2