Anti cd3e percp

After euthanasia, cell suspensions of spleen (at weaning and 5 days) and mesenteric lymphoid nods (only at weaning) were obtained by mechanically extrusion through a 40-μm nylon cell strainer (BD, Switzerland). Cells were washed through the strainer using 1 ml of Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, France) supplemented with 10% foetal bovine serum (FBS, Gibco). Erythrocytes were lysed by incubation with the red blood cell lysing buffer Hybri-Max (Sigma-Aldrich) according to manufacturer instructions. For each sample, aliquots of 10⁶ cells were transferred to two 96-well plates (Grenier, France). Following standard protocols as previously described [9 (link), 30 (link)], cells were stained with one of the next: (i) anti-CD4-FITC, anti-CD3e-percp and anti-T-bet-PE; (ii) anti-CD4-FITC, anti-CD3e-percp and anti-Gata3-PE; (iii) anti-CD4-FITC, anti-CD3e-percp and anti-rorγ-PE; or (iv) anti-CD4-FITC, anti-CD3e-percp and anti-Foxp3-PE (all from eBioscience, France). All stainings were performed in the presence of CD16/CD32 (eBioscience). Samples were subsequently analysed using an Accuri C6 cytometer (BD). The data obtained from the cytofluorimetric analysis were processed using CFlowSampler software (BD).

DPLN and spleens from PBS-inoculated or ECTV-infected BALB/c mice were collected at 2 and 7 dpi, respectively, in RPMI supplemented with 10% FCS. Cell suspensions were obtained by homogenization of the organs through 40 μm cell strainers (BD Bioscience). Red blood cells were lysed by hypoosmotic shock in milli-Q water and white cells were washed twice in PBS and counted manually in a haemocytometer. DPLN cells were stained with anti-DX5-FITC (eBioscience), anti-CD3e-PerCP (eBioscience) and anti-GzB-APC (R&D Biosystems). Splenocytes were stained with anti-CD3e-PerCP, anti-CD8-PE (eBioscience) and anti-GzB-APC. In parallel, cell suspensions were also stained with the appropriate isotype control antibodies. 100,000 cells were analyzed in a FACS Calibur flow cytometer (Becton Dickinson). Events were gated according to a forward and side scatter pattern compatible with healthy lymphocytes (Supplementary Fig. 3). Results were analyzed with FlowJo software (FlowJo LLC).