Flow Cytometry Profiling of CD4+ T Cells

Flow cytometry staining of markers on CD4⁺ T cells was performed on Cyto-Chex-stabilized whole blood [31 (link)]. Preanalysis was conducted to confirm that Cyto-Chex-stabilized blood generated data of all tested markers equivalent to that obtained with fresh blood samples (data not shown). Stainings of blood were adopted from protocols used for peripheral blood mononuclear cells [32 (link)]. Red blood cells were lyzed and remaining cells were stained with fluorochrome-conjugated antibodies (Supplemental Digital Content Table S2). Cells were permeabilized and fixed with the FOXP3 staining kit (eBioscience, San Diego, California, USA). Within 6 h, cells were run on a LSR Fortessa (BD Biosciences), where minimally 600 000 events were collected per run. Antibody capture beads (BD Biosciences) were used for compensation and FlowJo 8.8.7 (Tree Star, Ashland, Oregon, USA) for analyses. All manual gatings were based on fluorescence-minus-one gating strategies as described [33 (link),34 (link)]. A typical gating strategy to distinguish CD4⁺ T cells is visualized in Fig. 1a.