Daan Pieren

68 Chapter 3 Splenocyte culture and in vitro stimulation To investigate T-cell proliferation and upregulation of the activation marker CD25, splenic single cell suspensions were exposed to soluble anti-CD3 (0.019 µ g/mL; clone 145-2C11, eBioscience, San Diego, CA, USA) alone or in the presence of soluble anti-CD28 (0.5 µ g/mL; clone PV-1, Southern Biotech, Birmingham, AL, USA) or recombinant murine IL-2 (0.1 µ g/mL; eBioscience). Stimuli were prepared in RPMI-1640 medium containing 10% FCS and then added to splenocyte suspensions (4*10 5 cells/well). Cells were cultured in 96- well U-bottom plates (CELLSTAR, Greiner Bio-One) at 37°C and 5% CO 2 for four days. Immunofluorescence labeling and flow cytometric analyses Splenic single cell suspensions were washed with PBS containing 2% FCS and labeled for 30 minutes at 4°C for a combination of cell surface markers with the following fluorescently labeled anti-mouse antibodies: anti-CD4-BUV395 (clone GK1.5), anti-CD44-V450 (clone IM7) (BD Horizon, Franklin Lakes, NJ, USA); anti-CD25-PE-Cy7 (clone PC61.5) (eBioscience); CD122-PE-Dazzle 594 (clone TM-beta1), and anti-PD-1-BV785 (clone 29F.1A12) (BioLegend, San Diego, CA, USA). Live/Dead TM Fixable Aqua Dead Cell Stain Kit (Invitrogen) was included in the cell surface labeling to assess cell viability. Cells were subsequently labeled intracellularly according to the FoxP3 Transcription Factor staining buffer set protocol (eBioscience) with anti-CD3zeta-FITC (clone H146-968) (Abcam, Cambridge, Cambridgeshire, UK) and anti-FoxP3-eFluor660 (clone 150D/E4) (eBioscience). Labeled cells were detected on a BD LSRFortessa X-20 (BD Biosciences, Franklin Lakes, NJ, USA). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA). Dimensionality reduced analyses Dimensionality reduced analysis (viSNE) of flow cytometry data was performed in Cytobank (www.cytobank.com ) [32]. Cell density maps show clustering of CD4 + and CD4 - naive and memory T-cell populations that were generated from pooled flow cytometry datafiles of Ercc1 -/ Δ 7 mice (n=7) and pooled flow cytometry datafiles of the different Ercc1 +/+ mice (n=6). The number of cells included in the viSNE analysis were equal between Ercc1 -/ Δ 7 and Ercc1 +/+ mice; 2.3*10 5 naive CD4 + T cells, 1.4*10 5 naive CD4 - T cells, 3.3*10 4 memory CD4 + T cells, and 2.2*10 4 memory CD4 - T cells. Clustering was based on expression of FoxP3, CD25, CD122, and PD-1. Expression of the designated cellular markers in the heatmaps was based on their ArcSinh5-transformed median expression.