Immunomagnetic enrichment and flow cytometric characterization of mouse microglia

Abstract

Background: The 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 method: We 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(+)CD45(lo) microglia were evaluated by expression of M1 surface marker FcγRII/III and M2 surface marker CD206.

Results: After TBI, we observed an increase in M1:M2 ratio in the ipsilateral hemisphere when compared to the contralateral side, indicating that 24h 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.

Conclusions: In 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.

Keywords: Flow cytometry; Microglia; Neuroinflammation; Traumatic brain injury.

Fig. 1

Sequence and timing of steps for preparation of injured mouse brain hemispheres for flow cytometry.

Fig. 2

Percent myeloid and nonmyeloid leukocytes in unseparated and immunomagnetically enriched neural cell suspensions determined by flow cytometry. (A) CD11b⁺ myeloid cells (large plot, red box) and CD11b⁻CD45⁺ nonmyeloid leukocytes (blue box) were enumerated after any remaining myelin debris and aggregates were eliminated by exclusion gates based on scatter characteristics (small plots). (B) Ipsilateral injured hemispheres (CCI-R) showed significantly lower pre-enrichment frequencies of CD11b⁺ cells compared to contralateral hemispheres (CCI-L) and uninjured brains. (C) Increased frequencies of CD11b⁻CD45⁺ nonmyeloid leukocytes were observed in contralateral CCI-L versus ipsilateral CCI-R hemispheres. Purity (D) and fold enrichment (E) of CD11b-enriched cell suspensions also varied significantly in injured right hemispheres. * indicates p <0.05, ** indicates p <0.01, *** indicates p <0.005, and # indicates one-tailed p ≤ 0.05

Fig. 3

Two populations of CD11b⁺ myeloid cells in immunomagnetically enriched neural cell suspensions identified by flow cytometry. (A) Both CD11b⁺CD45^lo microglia (red box) and CD11b⁺CD45^hi cells (blue box) were evident after excluding debris, aggregates, and CD11b⁻ cells from the analysis (small plots). (B) Representative histograms display higher forward scatter (FSC-A) values and brighter CD11b and F_cγRII/III staining of CD11b⁺CD45^hi cells (blue lines) compared to the CD11b⁺CD45^lo microglial population (red lines). (C, D) The relative frequencies of these two myeloid populations varied significantly between ipsilateral CCI-R and contralateral CCI-L hemispheres 24 hours after CCI injury. * indicates p <0.05

Fig. 4

M1 and M2 microglia in immunomagnetically enriched neural cell suspensions identified by flow cytometry. (A) CD11b⁺CD45^lo microglia (large plot) were categorized as M1 (red box) or M2 (blue box) macrophage/microglia based on CD206 and F_cγRII/III expression after excluding debris, aggregates, CD11b⁻, and CD45^hi cells from the analysis (small plots). (B) Injured CCI-R hemispheres showed an increase in % M1 cells (p = 0.0565) compared to contralateral CCI-L hemispheres. (C) Injured CCI-R hemispheres also exhibited a significant decrease in % M2 cells compared to contralateral CCI-L hemispheres. (D) Correspondingly, the M1:M2 ratio, the number of M1 microglia divided by the number of M2 microglia, increased significantly in CCI-R compared to CCI-L hemispheres 24 hours after injury. * indicates p <0.05, # indicates one-tailed p ≤ 0.05