Lymphocyte Immunophenotyping at Weaning and Adolescence

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).