Immunomagnetic enrichment and flow cytometric characterization of mouse microglia.
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.


METHODS
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.
Department of Pediatric Surgery, University of Texas, Health Science Center at Houston, Houston, TX, United States.
Supinder S
SS
Bedi
Philippa
P
Smith
Robert A
RA
Hetz
Hasen
H
Xue
Charles S
CS
Cox
D000818:Animals; D001921:Brain / Q000473:pathology; D001930:Brain Injuries / Q000473:pathology; D039481:CD11b Antigen / Q000032:analysis; D002469:Cell Separation; D003627:Data Interpretation, Statistical; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D018189:Immunomagnetic Separation / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000032:analysis; D007958:Leukocyte Count; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000502:physiology*
D039481:CD11b Antigen; D005456:Fluorescent Dyes; D017493:Leukocyte Common Antigens
Flow cytometry; Microglia; Neuroinflammation; Traumatic brain injury