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

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.

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.

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.

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.