Cyan adp 9 colour analyser

Flow cytometry was performed using a CyAn ADP 9 colour analyser (Beckman Coulter, UK) equipped with 405 nm, 488 nm and 642 nm solid-state lasers and 11 detectors in standard configuration. Summit software was used for both acquisition and analysis (Beckman Coulter, UK). Prior to acquisition, the machine was calibrated with single peak alignment beads (Spherotech, USA), checking that coefficients of variation resided within the target ranges. Samples were filtered through 35 μm nylon cell strainer mesh tubes (BD Biosciences, UK) immediately prior to acquisition and a minimum of 250,000 events were acquired for each sample. Data analysis is described in the main text. In brief, events were first plotted forward versus side scatter using side scatter on a log scale and a large gate was drawn excluding debris. Events were then plotted for either CD3 or CD11c versus forward scatter area to identify CD3⁺ T cells and CD11c⁺ phagocytes. CD3⁺ and CD11c⁺ gated cells were then plotted versus the dead cell stain 7-AAD and a live (negatively staining) gate was drawn. Live CD3⁺ or CD11c⁺ gated cells were finally plotted either CD3⁺ (T cells) or CD11c⁺ (myeloid cells) versus side scatter (SSC log scale). A gate was drawn to identify SSC high cells based on the negative controls.