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Flow cytometric characterization of brain dendritic cell subsets after murine stroke.

Pösel C, Uri A, Schulz I, Boltze J, Weise G, Wagner DC - Exp Transl Stroke Med (2014)

Bottom Line: Sterile inflammation is a substantial element of post-stroke pathophysiology with the determination of autoimmunity versus tolerance being one of its most important aspects.The latter could be differentiated into the classical migratory DC subpopulations (CD11b+ and CD103+), but no CD4 or CD8+ DC were found.Finally, stroke caused a significant increase of CD11b/CD103 double-positive DC in the affected brain hemisphere.

View Article: PubMed Central - PubMed

Affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

ABSTRACT

Background: Sterile inflammation is a substantial element of post-stroke pathophysiology with the determination of autoimmunity versus tolerance being one of its most important aspects. It is believed that this determination is initiated relatively early after stroke onset by clearing macrophages and migratory dendritic cells (DC). However, the phenotypic differentiation of macrophages and DC is intricate particularly in the disease context. Here, we utilized a set of surface markers used in mucosal immunity research to investigate the involvement of macrophages and DC subpopulations in post-stroke inflammation in mice.

Findings: Photothrombotic stroke induced a significant increase of lineage (CD3, B220, Ly6G and CD49b) negative CD11b+ cells in the brain primarily consisting of F4/80+ macrophages and, to a lesser extent, F4/80-/CD11c-/CD11b+ monocytes and F4/80-/CD11c+ DC. The latter could be differentiated into the classical migratory DC subpopulations (CD11b+ and CD103+), but no CD4 or CD8+ DC were found. Finally, stroke caused a significant increase of CD11b/CD103 double-positive DC in the affected brain hemisphere.

Conclusions: The surface marker combination used in this study allowed a phenotypic differentiation of macrophages and DC subpopulations after stroke, thus providing an important prerequisite to study post-stroke immunity and tolerance.

No MeSH data available.


Related in: MedlinePlus

Gating strategy for the analysis of brain leukocytes. Lineage (Lin) antibodies include CD3 (T cells), B220 (B cells, pDC), Ly6G (neutrophils) and CD49b (NK cells, NKT cells). FSC-H, forward scatter height; FSC-A, forward scatter area; SSC-A, side scatter area; Ma, macrophages.
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Fig1: Gating strategy for the analysis of brain leukocytes. Lineage (Lin) antibodies include CD3 (T cells), B220 (B cells, pDC), Ly6G (neutrophils) and CD49b (NK cells, NKT cells). FSC-H, forward scatter height; FSC-A, forward scatter area; SSC-A, side scatter area; Ma, macrophages.

Mentions: Cell suspensions were first incubated with an anti-mouse CD16/CD32 Fc-receptor blocking reagent (eBioscience, Frankfurt, Germany) for 10min before being labeled with the following anti-mouse antibodies: F4/80-Alexa Fluor488 (AbD Serotec Kidlington, UK), CD4-PerCP, MHCII (I-A/I-E)-PerCP, CD11b-APC, CD45.2-APC-eFluor780 (all eBioscience), CD11c-PE-Cy7, CD103-Pacific Blue (both Biolegend, San Diego, USA), CD8-Horizon V500 (BD Biosciences, Heidelberg, Germany) and biotinylated anti-lineage antibodies (CD3, B220, Ly6G, CD49b, all ebioscience) followed by incubation with phycoerythrin-labeled streptavidin (BD Biosciences). Flow cytometric analyses were performed by an investigator blinded to the group allocation using a 3-laser FACSCanto II (BD Biosciences) and analyzed by FlowJo software (Tree Star, Ashland, USA). The gating strategy is displayed in Figure 1. Total brain leukocyte counts were determined by Trucount Tube measurement (BD Biosciences) of CD45-labeled brain cell suspensions.Figure 1


Flow cytometric characterization of brain dendritic cell subsets after murine stroke.

Pösel C, Uri A, Schulz I, Boltze J, Weise G, Wagner DC - Exp Transl Stroke Med (2014)

Gating strategy for the analysis of brain leukocytes. Lineage (Lin) antibodies include CD3 (T cells), B220 (B cells, pDC), Ly6G (neutrophils) and CD49b (NK cells, NKT cells). FSC-H, forward scatter height; FSC-A, forward scatter area; SSC-A, side scatter area; Ma, macrophages.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230520&req=5

Fig1: Gating strategy for the analysis of brain leukocytes. Lineage (Lin) antibodies include CD3 (T cells), B220 (B cells, pDC), Ly6G (neutrophils) and CD49b (NK cells, NKT cells). FSC-H, forward scatter height; FSC-A, forward scatter area; SSC-A, side scatter area; Ma, macrophages.
Mentions: Cell suspensions were first incubated with an anti-mouse CD16/CD32 Fc-receptor blocking reagent (eBioscience, Frankfurt, Germany) for 10min before being labeled with the following anti-mouse antibodies: F4/80-Alexa Fluor488 (AbD Serotec Kidlington, UK), CD4-PerCP, MHCII (I-A/I-E)-PerCP, CD11b-APC, CD45.2-APC-eFluor780 (all eBioscience), CD11c-PE-Cy7, CD103-Pacific Blue (both Biolegend, San Diego, USA), CD8-Horizon V500 (BD Biosciences, Heidelberg, Germany) and biotinylated anti-lineage antibodies (CD3, B220, Ly6G, CD49b, all ebioscience) followed by incubation with phycoerythrin-labeled streptavidin (BD Biosciences). Flow cytometric analyses were performed by an investigator blinded to the group allocation using a 3-laser FACSCanto II (BD Biosciences) and analyzed by FlowJo software (Tree Star, Ashland, USA). The gating strategy is displayed in Figure 1. Total brain leukocyte counts were determined by Trucount Tube measurement (BD Biosciences) of CD45-labeled brain cell suspensions.Figure 1

Bottom Line: Sterile inflammation is a substantial element of post-stroke pathophysiology with the determination of autoimmunity versus tolerance being one of its most important aspects.The latter could be differentiated into the classical migratory DC subpopulations (CD11b+ and CD103+), but no CD4 or CD8+ DC were found.Finally, stroke caused a significant increase of CD11b/CD103 double-positive DC in the affected brain hemisphere.

View Article: PubMed Central - PubMed

Affiliation: Fraunhofer Institute for Cell Therapy and Immunology, Leipzig, Germany.

ABSTRACT

Background: Sterile inflammation is a substantial element of post-stroke pathophysiology with the determination of autoimmunity versus tolerance being one of its most important aspects. It is believed that this determination is initiated relatively early after stroke onset by clearing macrophages and migratory dendritic cells (DC). However, the phenotypic differentiation of macrophages and DC is intricate particularly in the disease context. Here, we utilized a set of surface markers used in mucosal immunity research to investigate the involvement of macrophages and DC subpopulations in post-stroke inflammation in mice.

Findings: Photothrombotic stroke induced a significant increase of lineage (CD3, B220, Ly6G and CD49b) negative CD11b+ cells in the brain primarily consisting of F4/80+ macrophages and, to a lesser extent, F4/80-/CD11c-/CD11b+ monocytes and F4/80-/CD11c+ DC. The latter could be differentiated into the classical migratory DC subpopulations (CD11b+ and CD103+), but no CD4 or CD8+ DC were found. Finally, stroke caused a significant increase of CD11b/CD103 double-positive DC in the affected brain hemisphere.

Conclusions: The surface marker combination used in this study allowed a phenotypic differentiation of macrophages and DC subpopulations after stroke, thus providing an important prerequisite to study post-stroke immunity and tolerance.

No MeSH data available.


Related in: MedlinePlus