Isolation and Culture of Microglia

Abstract

Microglia represent 5-10% of cells in the central nervous system and contribute to the development, homeostasis, injury, and repair of neural tissues. As the tissue-resident macrophages of the central nervous system, microglia execute core innate immune functions such as detection of pathogens/damage, cytokine secretion, and phagocytosis. However, additional properties that are specific to microglia and their neural environment are beginning to be appreciated. This article describes approaches for purification of microglia by fluorescence-activated cell sorting using microglia-specific surface markers and for enrichment of microglia by magnetic sorting and immunopanning. Detailed information about culturing primary microglia at various developmental stages is also provided. Throughout, we focus on special considerations for handling microglia and compare the relative strengths or disadvantages of different protocols. © 2018 by John Wiley & Sons, Inc.

Keywords: FACS; MACS; culture; fluorescence-activated cell sorting; immunopanning; magnetic-activated cell sorting; microglia; tissue-resident macrophage.

Figure 1.. Representative gating strategy for mouse (top row) and human (bottom row) microglia sorting

We typically gate on FSC/SSC, then single cells, then live cells for both mouse and human. Depending on the goals of a given sort, we then either gate on CD45/CD11B double-positive populations, and individually sort TMEM119-positive and negative cells within this gate (as shown for human microglia), or gate directly on TMEM119-positive cells (as shown for mouse microglia, where a CD45/CD11B double-positive gate is shown, but not used in the gating hierarchy, as evidenced by the very large TMEM119-negative population in the right most column).

Figure 2.. Microglial morphology over six days in culture

Representative phase-contrast images of P14 rat microglial cultures over six days in culture illustrating morphological and proliferative differences between cells grown with serum-free medium (top) versus medium containing 10% fetal calf serum (FCS, bottom). CD11b-immunopanned cells were isolated as described in Alternate Protocol 2 and spot-plated as described in Basic Protocol 3 except that IL-34 (100 ng/mL) was used in place of CSF-1. The same field was imaged every 24 hours. Scale bar, 100 μm. This figure is duplicated from a previous work and used with permission (Collins & Bohlen, 2018).