Cell sorting was performed on a FACSAria W or L (BD Biosciences) cell sorter. For IL-2Rα and CD28 level sorting, cells were labelled with anti-CD28-PECy7 (clone 37.51, eBioscience) or anti-CD25-FITC (clone 7D4, BD).
Flow cytometry was performed on a FACSCanto II or LSRFortessa X-20 cytometer (both BD Biosciences). Data were analysed using FlowJo software (Treestar).
A known number of beads (Rainbow calibration particles, BD Biosciences) and propidium iodide (0.2 μg ml−1, Sigma) was added to samples immediately prior to analysis. The ratio of beads to live cells was used to estimate the absolute cell number. The following monoclonal antibodies were used for the detection of cell surface markers: anti-CD25 -PECy7, or—APC (clone PC61, BD Biosciences) anti-CD28-PECy7 (clone 37.51, eBioscience). Staining was performed in PBS containing 0.1% BSA and 0.1% sodium azide (Sigma). InSupplementary Fig. 8 , activated cells were defined as the 50% of cells with the highest FSC fluorescence. Spearman's correlation was calculated using Matlab 2011a's corr function.
Flow cytometry was performed on a FACSCanto II or LSRFortessa X-20 cytometer (both BD Biosciences). Data were analysed using FlowJo software (Treestar).
A known number of beads (Rainbow calibration particles, BD Biosciences) and propidium iodide (0.2 μg ml−1, Sigma) was added to samples immediately prior to analysis. The ratio of beads to live cells was used to estimate the absolute cell number. The following monoclonal antibodies were used for the detection of cell surface markers: anti-CD25 -PECy7, or—APC (clone PC61, BD Biosciences) anti-CD28-PECy7 (clone 37.51, eBioscience). Staining was performed in PBS containing 0.1% BSA and 0.1% sodium azide (Sigma). In