MLNL phenotypic analysis

MLNLs (5 × 10⁵ cells) were extracellularly and intracellularly stained by using mouse anti-rat monoclonal antibodies (mAb) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin-chlorophyll-a protein (PercP), allophycocyanin (APC) and brilliant-violet 421 (BV421), as previously described⁷². The following fluorochrome-conjugated mAb antibodies were used: FITC-TCRαβ, FITC-CD8β, FITC-CD25, PE-CD161a, PE-TCRγδ, PE-CD4, PerCP-CD8α, APC-CD4, and BV421-CD45RA (BD Biosciences, Madrid, Spain) and APC-FoxP3 (eBioscience, Frankfurt, Germany). For extracellular staining, MLNL were incubated with saturating amounts of mAb in PBS containing 2% FBS and 0.1% NaN₃ (darkness, 4 °C, 20 min). For intracellular staining, cells previously labelled extracellularly with anti-CD4-PE and anti-CD25-FITC mAb were treated with Foxp3 fixation/permeabilization kit (eBioscience). Then, intracellular staining with anti-Foxp3-APC mAb was carried out (darkness, 4 °C, 30 min), as described in previous studies⁷⁵. All stained cells were fixed with 0.5% p-formaldehyde and stored at 4 °C in darkness until analysis by flow cytometry. A negative control staining without any mAb antibody and a staining control for each mAb were included. Analyses were performed using a Gallios Cytometer (Beckman Coulter, Miami, FL, USA) in the Flow Cytometry Unit of the Scientific and Technological Centres of the University of Barcelona (CCiT-UB) and by Flowjo v10 software (Tree Star, Inc., Ashland, OR, USA). Changes in lymphocyte phenotype by exercise are represented considering the SED group mean value as 1, therefore, all values are expressed as a fold change of the mean value with respect to the SED group.