Purification of Resting Macrophage Subsets

All cells were purified using the sorting protocol and antibodies listed on http://www.immgen.org. Cells were directly sorted from mouse tissues and were processed from tissue procurement to a second round of sorting into Trizol within 4 h using a Beckton-Dickinson Aria II instrument. Resting red pulp macrophages from the spleen were sorted after nonenzymatic disaggregation of the spleen and were identified as F4/80^hi cells that lacked B220 and high levels of CD11c and MHC II^{34 (link),35 (link)}; macrophages from the resting peritoneum were collected in a peritoneal lavage and stained to identify CD115^hi cells that were F4/80^hi MHC II⁻; resting pulmonary macrophages were isolated from Liberase III-digested lungs (15 min. digest) and macrophages were identified as SiglecF⁺ CD11c⁺ cells with low levels of MHC II^{26 (link),27 (link)}; and resting brain microglial macrophages were sorted from Liberase III-digested, Percoll-gradient separated cells that were CD11b⁺ CD45^lo F4/80^{lo11 (link)}. The Data Browser in the Immgen website is a resource for pdf files showing FACS dot plots that depict the purification strategies and purity after isolation of these and all other populations. A list of abbreviations used in the Immgen database relevant to macrophages and DCs can be found in Supplementary Note 1.