![]() combined magnetic bead isolation techniques with cell sorting strategies to isolate RGCs with higher purity 16. 15 adapted the classic immunopanning technique and added a second step, which excluded monocytes and endothelial cells from the bulk of retinal cells prior to positive selection based upon immunopositivity to anti-thymocyte antigen (aka Thy1), a cell-surface marker. Early RGC isolation protocols were based on immunopanning methods. Finally, currently available protocols are lengthy and lack standardization 13, 14. Selection based upon these markers leaves the cells non-viable, which precludes downstream functional analyses. Second, most RGC-specific markers are intracellular proteins 10, 11, 12. RGCs account for less than 1% of total retinal cells 7, 8, 9. First, the number of RGCs present in a murine retina is small. A series of challenges impede progress towards the use of these cells for in-depth studies. Lack of oxygenation at the optic nerve head 1, 2, 3 causes RGC death 4 and acts as the disturbance of the homeostasis between the activation of excitatory and inhibitory receptors within individual RGCs 5, 6. Although the specific cellular mechanisms underlying RGC loss are unclear, a series of risk factors have been identified. The degeneration of RGCs is associated with retinal degenerative diseases, such as glaucoma, diabetic retinopathy, and normal aging. This is especially important for studies that seek to generate potential therapies to promote RGC function and to minimize their death. The development of a protocol for the isolation and enrichment of primary murine retinal ganglion cells (RGCs) is fundamental to revealing the mechanisms of RGC health and degeneration in vitro. RGCs are terminally differentiated neurons, and therefore, primary cells are required for experimentation. The presented methods allow for the future study of RGCs, with the goal of better understanding the major decline in visual acuity that results from the loss of functional RGCs in neurodegenerative diseases. This work describes an efficient, comprehensive, and reliable method to isolate primary RGCs from mice retinae using a protocol based on both positive and negative selection criteria. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. Despite their importance, RGCs have been extremely difficult to study until now due in part to the fact that they comprise only a small percentage of the wide variety of cells in the retina. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. Neurodegenerative diseases often have a devastating impact on those affected. ![]()
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