Flow cytometric analysis of leukocytes in brain
Mice were killed by carbon dioxide inhalation and perfused through the left cardiac ventricle with 0.2 % clexane (400 IU, Sanofi Aventis, Australia) in 0.01 M phosphate-buffered saline (PBS). The left brain hemisphere was harvested for flow cytometry. Brain samples were minced with scissors and digested in PBS (with MgCl2 and CaCl2) containing a mixture of collagenase type XI (125 U/ml), collagenase type I-S (460 U/ml) and hyaluronidase (60 U/ml) (Sigma-Aldrich, USA) for 30 min at 37 °C. Brain samples were then passed through a 70 μm filter and subjected to a PercollTMgradient (70 % and 30 % isotonic Percoll) centrifugation from which the layer of mononuclear cells was collected from the interface of the Percoll solutions. Brain cells were stained with an antibody cocktail of anti-CD45 APC-Cy7 (30-F11, Biolegend, USA), anti-CD11b Pacific Blue (M1-70, eBioscience, USA), anti-Ly6G PE-Cy7 (1A8, Biolegend, USA), anti-Ly6C FITC (HK1.4, Biolegend, USA), anti-CD3ε APC (145-2C11, eBioscience, USA), anti-CD4 Alexa Fluor 700 (GK1.5, eBioscience, USA), anti-F4/80 BV711 (BM8, Biolegend, USA) and anti-CD19 PE-Cy5 (6D5, Biolegend, USA), and diluted in PBS containing 0.5% bovine serum albumin. Samples were analysed via flow cytometry using a CytoFLEX LX flow cytometer (Beckman Coulter, USA) and FlowJo Software (version 10.1, Tree Star Inc, USA, Supplementary Figure 1). Cell numbers were expressed as total cells per brain hemisphere.