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Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry.

Portillo JA, Okenka G, Kern TS, Subauste CS - Mol. Vis. (2009)

Bottom Line: Costaining with antibodies against intercellular adhesion molecule-1 (ICAM-1) and CD40 revealed that ICAM-1 is normally expressed at various levels on all subsets of retinal cells examined.In contrast, CD40 was detected only on CD11b(+), CD31(+), Thy-1(+), and vimentin(+) cells.Ischemia-reperfusion of the retina resulted in upregulation of ICAM-1 on CD105(+) and vimentin(+) cells and upregulation of nitric oxide synthase 2 in CD11b(+) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.

ABSTRACT

Purpose: Advances in the understanding of the pathogenesis of retinal disorders can be facilitated by a methodology to measure expression of proinflammatory molecules in various subsets of retinal cells.

Methods: We examined whether a multiparameter flow cytometric assay can be used to identify various subsets of retinal cells and examine expression of molecules involved in inflammatory responses in the retina. Single-cell suspensions freshly obtained after enzymatic digestion of normal mouse retinas were stained with antibodies against cluster of differentiation 11b (CD11b), cluster of differentiation 31 (CD31), Glial fibrillary acidic protein (GFAP), rhodopsin, Thy-1, and vimentin. These markers were previously shown by immunohistochemistry to label retinal microglia/macrophages, endothelial cells, astrocytes, photoreceptors, ganglion neurons, and Müller cells respectively in normal mouse retinas.

Results: Costaining with antibodies against intercellular adhesion molecule-1 (ICAM-1) and CD40 revealed that ICAM-1 is normally expressed at various levels on all subsets of retinal cells examined. In contrast, CD40 was detected only on CD11b(+), CD31(+), Thy-1(+), and vimentin(+) cells. Ischemia-reperfusion of the retina resulted in upregulation of ICAM-1 on CD105(+) and vimentin(+) cells and upregulation of nitric oxide synthase 2 in CD11b(+) cells.

Discussion: These results indicate that flow cytometry can be used to readily quantitate expression of surface and intracellular molecules of relevance to retinopathies in freshly isolated retinal cells.

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Expression of CD40 on various subsets of retinal cells. Single-cell suspensions were stained with isotype control or with anti-CD40 mAb. Cells were coincubated with antibodies that detect cell surface expression of CD11b, CD31, CD105, or Thy-1 (A), or cells were permeabilized followed by incubation with antibodies against rhodopsin and vimentin (B), or GFAP (C) to detect intracellular expression of these markers. PE-conjugated anti-CD40 mAb was used in all conditions except when staining GFAP+ cells, where FITC-conjugated mAb was used instead. The average expression of CD40 (cMFI) on gated CD11b+, CD31+, CD105+, Thy-1+, GFAP+, rhodopsin+, and vimentin+ events were calculated as described in Methods. Data are shown as the mean±SEM from three to four independent experiments. CD40 was not detected on rhodopsin+ and GFAP+ cells. D: Representative dot plots of CD40 versus various markers are shown. Red dots represent cells that expressed these markers. Horizontal and vertical lines represent gates obtained with isotype control antibodies.
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f4: Expression of CD40 on various subsets of retinal cells. Single-cell suspensions were stained with isotype control or with anti-CD40 mAb. Cells were coincubated with antibodies that detect cell surface expression of CD11b, CD31, CD105, or Thy-1 (A), or cells were permeabilized followed by incubation with antibodies against rhodopsin and vimentin (B), or GFAP (C) to detect intracellular expression of these markers. PE-conjugated anti-CD40 mAb was used in all conditions except when staining GFAP+ cells, where FITC-conjugated mAb was used instead. The average expression of CD40 (cMFI) on gated CD11b+, CD31+, CD105+, Thy-1+, GFAP+, rhodopsin+, and vimentin+ events were calculated as described in Methods. Data are shown as the mean±SEM from three to four independent experiments. CD40 was not detected on rhodopsin+ and GFAP+ cells. D: Representative dot plots of CD40 versus various markers are shown. Red dots represent cells that expressed these markers. Horizontal and vertical lines represent gates obtained with isotype control antibodies.

Mentions: ICAM-1 is an adhesion molecule that plays a pivotal role in recruitment of leukocytes to the retina and the development of vascular histopathology [2,5]. In addition, recent studies identified CD40 as an important regulator of retinal inflammation and neurovascular degeneration [16]. We used flow cytometry to examine the expression of these molecules on various retinal cells. Cell suspensions were stained with anti-ICAM-1 or anti-CD40 mAb plus either antibodies against CD11b, CD31, CD105, GFAP, rhodopsin, Thy-1, or vimentin. Varying levels of ICAM-1 were detected on all the subsets of retinal cells examined (Figure 3). ICAM-1 expression did not appear to be homogenous within rhodopsin+ and Thy-1+ cell populations. CD40 was detected only on CD11b+ (microglia/macrophages), CD31+(endothelial), Thy-1+(ganglion), and vimentin+ cells (Müller) cells (Figure 4). Despite the typical low levels of expression, CD40 is an important mediator of inflammation [15]. These results indicate that flow cytometry allows quantitation of surface molecules involved in retinal inflammatory disorders.


Identification of primary retinal cells and ex vivo detection of proinflammatory molecules using flow cytometry.

Portillo JA, Okenka G, Kern TS, Subauste CS - Mol. Vis. (2009)

Expression of CD40 on various subsets of retinal cells. Single-cell suspensions were stained with isotype control or with anti-CD40 mAb. Cells were coincubated with antibodies that detect cell surface expression of CD11b, CD31, CD105, or Thy-1 (A), or cells were permeabilized followed by incubation with antibodies against rhodopsin and vimentin (B), or GFAP (C) to detect intracellular expression of these markers. PE-conjugated anti-CD40 mAb was used in all conditions except when staining GFAP+ cells, where FITC-conjugated mAb was used instead. The average expression of CD40 (cMFI) on gated CD11b+, CD31+, CD105+, Thy-1+, GFAP+, rhodopsin+, and vimentin+ events were calculated as described in Methods. Data are shown as the mean±SEM from three to four independent experiments. CD40 was not detected on rhodopsin+ and GFAP+ cells. D: Representative dot plots of CD40 versus various markers are shown. Red dots represent cells that expressed these markers. Horizontal and vertical lines represent gates obtained with isotype control antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713733&req=5

f4: Expression of CD40 on various subsets of retinal cells. Single-cell suspensions were stained with isotype control or with anti-CD40 mAb. Cells were coincubated with antibodies that detect cell surface expression of CD11b, CD31, CD105, or Thy-1 (A), or cells were permeabilized followed by incubation with antibodies against rhodopsin and vimentin (B), or GFAP (C) to detect intracellular expression of these markers. PE-conjugated anti-CD40 mAb was used in all conditions except when staining GFAP+ cells, where FITC-conjugated mAb was used instead. The average expression of CD40 (cMFI) on gated CD11b+, CD31+, CD105+, Thy-1+, GFAP+, rhodopsin+, and vimentin+ events were calculated as described in Methods. Data are shown as the mean±SEM from three to four independent experiments. CD40 was not detected on rhodopsin+ and GFAP+ cells. D: Representative dot plots of CD40 versus various markers are shown. Red dots represent cells that expressed these markers. Horizontal and vertical lines represent gates obtained with isotype control antibodies.
Mentions: ICAM-1 is an adhesion molecule that plays a pivotal role in recruitment of leukocytes to the retina and the development of vascular histopathology [2,5]. In addition, recent studies identified CD40 as an important regulator of retinal inflammation and neurovascular degeneration [16]. We used flow cytometry to examine the expression of these molecules on various retinal cells. Cell suspensions were stained with anti-ICAM-1 or anti-CD40 mAb plus either antibodies against CD11b, CD31, CD105, GFAP, rhodopsin, Thy-1, or vimentin. Varying levels of ICAM-1 were detected on all the subsets of retinal cells examined (Figure 3). ICAM-1 expression did not appear to be homogenous within rhodopsin+ and Thy-1+ cell populations. CD40 was detected only on CD11b+ (microglia/macrophages), CD31+(endothelial), Thy-1+(ganglion), and vimentin+ cells (Müller) cells (Figure 4). Despite the typical low levels of expression, CD40 is an important mediator of inflammation [15]. These results indicate that flow cytometry allows quantitation of surface molecules involved in retinal inflammatory disorders.

Bottom Line: Costaining with antibodies against intercellular adhesion molecule-1 (ICAM-1) and CD40 revealed that ICAM-1 is normally expressed at various levels on all subsets of retinal cells examined.In contrast, CD40 was detected only on CD11b(+), CD31(+), Thy-1(+), and vimentin(+) cells.Ischemia-reperfusion of the retina resulted in upregulation of ICAM-1 on CD105(+) and vimentin(+) cells and upregulation of nitric oxide synthase 2 in CD11b(+) cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.

ABSTRACT

Purpose: Advances in the understanding of the pathogenesis of retinal disorders can be facilitated by a methodology to measure expression of proinflammatory molecules in various subsets of retinal cells.

Methods: We examined whether a multiparameter flow cytometric assay can be used to identify various subsets of retinal cells and examine expression of molecules involved in inflammatory responses in the retina. Single-cell suspensions freshly obtained after enzymatic digestion of normal mouse retinas were stained with antibodies against cluster of differentiation 11b (CD11b), cluster of differentiation 31 (CD31), Glial fibrillary acidic protein (GFAP), rhodopsin, Thy-1, and vimentin. These markers were previously shown by immunohistochemistry to label retinal microglia/macrophages, endothelial cells, astrocytes, photoreceptors, ganglion neurons, and Müller cells respectively in normal mouse retinas.

Results: Costaining with antibodies against intercellular adhesion molecule-1 (ICAM-1) and CD40 revealed that ICAM-1 is normally expressed at various levels on all subsets of retinal cells examined. In contrast, CD40 was detected only on CD11b(+), CD31(+), Thy-1(+), and vimentin(+) cells. Ischemia-reperfusion of the retina resulted in upregulation of ICAM-1 on CD105(+) and vimentin(+) cells and upregulation of nitric oxide synthase 2 in CD11b(+) cells.

Discussion: These results indicate that flow cytometry can be used to readily quantitate expression of surface and intracellular molecules of relevance to retinopathies in freshly isolated retinal cells.

Show MeSH
Related in: MedlinePlus