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

[摘要] Purpose: Advances in the understandingof the pathogenesis of retinal disorders can be facilitated by amethodology to measure expression of proinflammatory molecules invarious subsets of retinal cells. Methods: We examined whether amultiparameter flow cytometric assay can be used to identify varioussubsets of retinal cells and examine expression of molecules involvedin inflammatory responses in the retina. Single-cell suspensionsfreshly obtained after enzymatic digestion of normal mouse retinas werestained 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 previouslyshown by immunohistochemistry to label retinal microglia/macrophages,endothelial cells, astrocytes, photoreceptors, ganglion neurons, andMüller cells respectively in normal mouse retinas. Results: Costaining with antibodiesagainst intercellular adhesion molecule-1 (ICAM-1) and CD40 revealedthat ICAM-1 is normally expressed at various levels on all subsets ofretinal 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-1on CD105+ and vimentin+ cells and upregulation ofnitric oxide synthase 2 in CD11b+ cells. Discussion: These results indicate thatflow cytometry can be used to readily quantitate expression of surfaceand intracellular molecules of relevance to retinopathies in freshlyisolated retinal cells.

[学科分类] 生物化学/生物物理