Immunomagnetic enrichment and flow cytometric characterization of mouse microglia

• We used a controlled cortical impact mouse model for traumatic brain injury.
• We immunomagnetically enriched brain cell suspensions for myeloid immune cells.
• We stained surface antigens to identify microglia flow cytometrically as M1 or M2.
• These methods allow quantitative measurement of microglial polarization states.
• 24 h after TBI, microglial M1:M2 ratio increased in injured brain hemispheres.

BackgroundThe inflammatory response after a CNS injury is regulated by microglia/macrophages. Changes in the ratio of M1 classically activated pro-inflammatory cells versus M2 alternatively activated anti-inflammatory cells reveal the direction of the immune response. These cells are routinely identified and enumerated by morphology and cell-surface markers using immunohistochemistry.New methodWe used a controlled cortical impact (CCI) mouse model for traumatic brain injury (TBI), then isolated microglia/macrophages from neural cell suspensions using magnetic beads conjugated to CD11b monoclonal antibody to obtain the entire myeloid population. Polarization states of CD11b+CD45lo microglia were evaluated by expression of M1 surface marker FcγRII/III and M2 surface marker CD206.ResultsAfter TBI, we observed an increase in M1:M2 ratio in the ipsilateral hemisphere when compared to the contralateral side, indicating that 24 h after CCI, a shift in microglia polarization occurs localized to the hemisphere of injury. Comparison with existing method(s): The major impetus for developing and refining the methods was the need to accurately quantify microglial activation states without reliance on manual morphometric counting of serial immunohistochemistry slides. Flow cytometric analysis of enriched cell suspensions provides quantitative measurement of microglial polarization states complementary to existing methods, but for entire populations of cells.ConclusionsIn summary, we used immunomagnetic beads to isolate myeloid cells from injured brain, then stained surface antigens to flow cytometrically identify and categorize microglia as either classically activated M1 or alternatively activated M2, generating a ratio of M1:M2 cells which is useful in studying attempts to reduce or redirect neuroinflammation.