Comprehensive Flow Cytometric Analysis

For flow cytometric analysis, splenocytes (2 × 10⁵) were labelled with mouse anti-rat monoclonal antibodies (mAb) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridininchlorophylla protein (PercP), allophycocyanin (APC) or APC-Cyanine (Cy)7, as in previous studies^{60 (link)}. In this case, the antibodies used were anti-CD4, anti-CD8α, anti-CD8β, anti-TCRαβ, anti-TCRγδ, anti-NKR-P1A, anti-CD25, anti-CD45RA (BD Biosciences, San Diego, USA), anti-CD62L, anti-CD103 (Biolegend, San Diego, CA, USA), anti-TLR-4 (Novus Biologicals, Littlon, CO, USA) and anti-Foxp3 (eBioscience). After staining with standard procedures^{20 (link)}, analyses were performed using a Gallios^TM Cytometer (Beckman Coulter Inc., Miami, FL, USA) at the CCiT-UB. All results were assessed by the Flowjo v.10 software (TreeStar, Inc., Ashland, OR, USA).