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


CONCLUSIONS
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.
Portillo
Jose-Andres C
JA
Department of Ophthalmology and Visual Sciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Okenka
Genevieve
G
Kern
Timothy S
TS
Subauste
Carlos S
CS
D000818:Animals; D019013:CD40 Antigens / Q000378:metabolism; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D018836:Inflammation Mediators / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000201:enzymology / Q000473:pathology; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010641:Phenotype; D015427:Reperfusion Injury / Q000201:enzymology / Q000473:pathology; D012160:Retina / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology*; D015854:Up-Regulation / Q000235:genetics; D014746:Vimentin / Q000378:metabolism
D019013:CD40 Antigens; D018836:Inflammation Mediators; D014746:Vimentin; D018799:Intercellular Adhesion Molecule-1; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse