Multiparametric Immune Cell Profiling by Flow Cytometry

MLNLs (500,000 cells/tube) were stained using mouse anti-rat monoclonal antibodies conjugated to FITC, phycoerythrin (PE), peridinin-chlorophyll-protein (PerCP), allophycocyanin (APC) or APC-cyanine (Cy)7. The antibodies used herein were anti-TCRαβ, anti-CD8α, anti-CD4, anti-TCRγδ and anti-CD45RA (BD Biosciences, San Diego, CA, USA). Cells were mixed with PBS containing 2% FBS and 1% NaN₃ and stained, as previously described [28 (link)]. The data were acquired with a Gallios™ Cytometer (Beckman Coulter, Miami, FL, USA) in the CCiT-UB and assessed by the Flowjo v10 software (Tree Star, Inc., Ashland, OR, USA). Results are expressed as percentages of positive cells in the lymphocyte population, selected according to their forward-scatter characteristics (FSC) and side-scatter characteristics (SSC).