Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Abstract

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11bCD45) from the MDM (CD11bCD45). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

Selective role of Na+/H+ exchanger in Cx3cr1+ microglial activation, white matter demyelination, and post‐stroke function recovery

“…A very small population of cells expressing CD45 high are present in the normal CNS, which normally represent as infiltrating macrophages (Sedgwick et al, 1991). As reported previously (Christensen etal., 2014; Martin, El-Behi, Fontaine, & Delarasse, 2017; Ritzel et al, 2015; Wong et al, 2017), CD11b + / CD45 low-med microglia and CD11b + /CD45 hi macrophage populations were identified in a scatter plot (Figure 2a). Supporting Information Figure 3 shows that these microglial populations also expressed P2RY12, a special parenchymal microglial marker protein (Butovsky et al, 2014; Mildner, Huang, Radke, Stenzel, & Priller, 2017), with a strong positive correlation (a Pearson coefficient r of .9638) between CD11b + /CD45 low-med and P2RY12 + cell counts (Supporting Information Figure 3b).…”

Schwann Cell Transplantation Subdues the Pro-Inflammatory Innate Immune Cell Response after Spinal Cord Injury

“…Control cell suspensions (unstained and single-color control) were employed for setting compensation. The gating strategy used for the selection of infiltrating macrophage and resident microglial populations was based upon the forward (size) and the side scatter (granularity) of these innate immune cells to eliminate any dead cells or debris in combination with the expression of markers, CD11b-conjugated to Pacific Blue or CD68-conjugated to Phycoerythrin, routinely used to specifically identify macrophages and microglia [ 60 ]. To further resolve the phenotype of these CD11b or CD68 innate immune cell populations, additional antibodies (iNOS-FITC and CD38-APC or CD163-FITC and Arg1-APC) were employed and percentages of CD11b or CD68 cells expressing multiple markers following quadrant analysis were calculated and averaged across animals.…”

Gradual Increase in Environmental Light Intensity Induces Oxidative Stress and Inflammation and Accelerates Retinal Neurodegeneration

“…The immunogenicity against CD45 was also analyzed as a marker of the microglia and leucocyte population, which is increased in activated cells, infiltrating monocytes and macrophages. 38,41,47 Each mouse retina was analyzed individually, and at least three retinas from different animals for each experimental group were assessed. Data were acquired on an LSR Fortessa cytometer (BD Biosciences) and analyzed using FCS Express 6 flow cytometry software (De Novo, Los Angeles, CA, USA).…”