Dataset Information

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 Fc?RII/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.

SUBMITTER: Bedi SS

PROVIDER: S-EPMC3809889 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Publications

Immunomagnetic enrichment and flow cytometric characterization of mouse microglia.

Bedi Supinder S SS Smith Philippa P Hetz Robert A RA Xue Hasen H Cox Charles S CS

Journal of neuroscience methods 20130806 1

<h4>Background</h4>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.<h4>New method</h4>We used a controlled cortical impact (CCI) mouse model for traumatic brain injury (TB ...[more]

PMID: 23928152

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Project description:IntroductionScientific literature regarding the characterization of lymphocyte subpopulations of the cecal appendix is sparse, with few precedents limited to immunohistochemical techniques.MethodsWe conducted a prospective pilot study to characterize lymphocyte subpopulations of the cecal appendix in children. Participants were divided into three groups: (1) patients without histological acute appendiceal inflammation, (2) patients with histological uncomplicated acute appendicitis, and (3) patients with histological complicated acute appendicitis (gangrenous, perforated). A fresh sample of the base of the appendix was taken from all patients and a flow cytometric study was performed. Quantitative variables were compared using Kruskal-Wallis test and Mann-Whitney U test.ResultsThis study included 57 patients divided into Group 1 (n = 5), Group 2 (n = 37), and Group 3 (n = 15). Median values (IQR) of the percentage of B-lymphocytes were 67.8 [66.8-68.1] in group 1, 61.15 [53.74-66.4] in group 2, and 52.1 [33-62.02] in group 3 (p = 0.02). Median values (IQR) of the percentage of NK-lymphocytes were 0.26 [0.2-0.3] in group 1, 0.55 [0.37-0.66] in group 2, and 0.84 [0.35-1.45] in group 3 (p = 0.008). Median values (IQR) of the percentage of T-lymphocytes were 31.9 [31.7-33.1] in group 1, 37.68 [32.15-45.69] in group 2, and 46.9 [37.03-67] in group 3 (p = 0.02). Pair comparisons of groups 2 and 3 also showed significant differences in the percentage of B lymphocytes (p = 0.03) and NK-lymphocytes (p = 0.02).ConclusionsSignificant differences in lymphocyte subpopulations were identified according to the histologic grade of the cecal appendix. More specifically, a lower percentage of B-lymphocytes and a higher percentage of T- and NK-lymphocytes were observed in cases of acute appendicitis. These findings must be confirmed and their etiopathogenic, diagnostic, and prognostic implications elucidated in future studies with larger sample sizes.

Dataset Information

Immunomagnetic enrichment and flow cytometric characterization of mouse microglia.

Background

New method

Results

Conclusions

Publications

Immunomagnetic enrichment and flow cytometric characterization of mouse microglia.

Similar Datasets

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