Generation of Microglial Primary Cultures

For generation of microglial primary cultures, P0 WT or Spp1^KO/KO mice were decapitated, the brain was dissected from the skull and meninges were removed in ice-cold HBSS with 5% FBS. Eight to ten mice were pooled per culture preparation. Tissue was homogenized first with 2 ml pipettes (15 strokes) in 15 ml falcon tube and subsequently transferred to a prewet 50 ml tube with 70 μM strainer. The 15 ml tube was washed with HBSS and then put through filter to ensure all tissues were collected. The supernatant was removed and the cell pellet was resuspended in ice-cold 35% isotonic percoll. The interface was carefully created with HBSS. The samples were centrifuged for 40 min at 4 °C at 2,800g with no break and with slow acceleration and deceleration. The myelin layer and supernatant layers were aspirated, and the cell pellet was washed in HBSS. Cells were centrifuged and resuspended in 1 ml microglial media (DMEM F12 (Gibco), 5% FBS (Gibco), 1% pen-strep (Gibco), 50 ng ml⁻¹ CSF1 416-ML-010/CF (RnD Systems), 50 ng ml⁻¹ TGFb1 7666-MB-005/CF (RnD Systems) and 100 ng ml⁻¹ CX3CL1 472-FF-025/CF (RnD Systems)) for cell counting. Cells were used within 7–10 d of plating.