Name: isolation of primary murine retinal ganglion cells (rgcs) by flow cytometry
Uploaded: 2017-07-05T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry at JoVE.com

The authors would like to thank Mr. Tim Higgins, Senior Illustrator from the Department of Microbiology, Immunology and Biochemistry, for technical video assistance; Dr. Matthew W. Wilson for discussions and the members of the Jablonski and Morales-Tirado laboratories for their helpful comments. This work was supported by the Alcon Research Institute Young Investigator Award (VMM-T), the University of Tennessee Research Foundation (VMM-T), the National Eye Institute EY021200 (MMJ), the Gerwin Fellowship (VMM-T); the Gerwin Pre-doctoral Fellowship (ZKG), the Department of Defense Army Medical Research and Materiel Command (VMM-T), and the Unrestricted Grant from Research to Prevent Blindness.

All procedures detailed in the following protocol were approved by the Institutional Animal Care and Use Committee (IACUC) review board at the University of Tennessee Health Science Center (UTHSC) and followed the Association for Research in Vision and Ophthalmology (ARVO) Statements for the Use of Animals in Ophthalmic and Vision Research, in addition to the guidelines for laboratory animal experiments (Institute of Laboratory Animal Resources, Public Health Service Policy on Humane Care and Use of Laboratory Animals).<...

<ol>
	<li>Flammer, J., Orgul, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+nerve+blood-flow+abnormalities+in+glaucoma.">Optic nerve blood-flow abnormalities in glaucoma.</a> Prog Retin Eye Res. 17 (2), 267-289 (1998).</li><li>Bathija, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optic+nerve+blood+flow+in+glaucoma.">Optic nerve blood flow in glaucoma.</a> Clin Exp Optom. 83 (3), 180-184 (2000).</li><li>Osborne, N. N., Melena, J., Chidlow, G., Wood, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hypothesis+to+explain+ganglion+cell+death+caused+by+vascular+insults+at+the+optic+nerve+head:+possible+implication+for+the+treatment+of+glaucoma.">A hypothesis to explain ganglion cell death caused by vascular insults at the optic nerve head: possible implication for the treatment of glaucoma.</a> Br J Ophthalmol. 85 (10), 1252-1259 (2001).</li><li>Morgan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulation+and+axonal+transport+in+the+optic+nerve.">Circulation and axonal transport in the optic nerve.</a> Eye (Lond). 18 (11), 1089-1095 (2004).</li><li>Tian, N., Hwang, T. N., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+excitatory+and+inhibitory+spontaneous+synaptic+activity+in+mouse+retinal+ganglion+cells.">Analysis of excitatory and inhibitory spontaneous synaptic activity in mouse retinal ganglion cells.</a> J Neurophysiol. 80 (3), 1327-1340 (1998).</li><li>Schmidt, K. G., Bergert, H., Funk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+diseases+of+the+retina+and+potential+for+protection+and+recovery.">Neurodegenerative diseases of the retina and potential for protection and recovery.</a> Curr Neuropharmacol. 6 (2), 164-178 (2008).</li><li>Dreher, B., Sefton, A. J., Ni, S. Y., Nisbett, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology,+number,+distribution+and+central+projections+of+Class+I+retinal+ganglion+cells+in+albino+and+hooded+rats.">The morphology, number, distribution and central projections of Class I retinal ganglion cells in albino and hooded rats.</a> Brain Behav Evol. 26 (1), 10-48 (1985).</li><li>Williams, R. W., Strom, R. C., Rice, D. S., Goldowitz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+environmental+control+of+variation+in+retinal+ganglion+cell+number+in+mice.">Genetic and environmental control of variation in retinal ganglion cell number in mice.</a> J Neurosci. 16 (22), 7193-7205 (1996).</li><li>Jeon, C. J., Strettoi, E., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+cell+populations+of+the+mouse+retina.">The major cell populations of the mouse retina.</a> J Neurosci. 18 (21), 8936-8946 (1998).</li><li>Surgucheva, I., Weisman, A. D., Goldberg, J. L., Shnyra, A., Surguchov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-synuclein+as+a+marker+of+retinal+ganglion+cells.">Gamma-synuclein as a marker of retinal ganglion cells.</a> Mol Vis. 14, 1540-1548 (2008).</li><li>Nadal-Nicolas, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brn3a+as+a+marker+of+retinal+ganglion+cells:+qualitative+and+quantitative+time+course+studies+in+naive+and+optic+nerve-injured+retinas.">Brn3a as a marker of retinal ganglion cells: qualitative and quantitative time course studies in naive and optic nerve-injured retinas.</a> Invest Ophthalmol Vis Sci. 50 (8), 3860-3868 (2009).</li><li>Kwong, J. M., Caprioli, J., Piri, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+binding+protein+with+multiple+splicing:+a+new+marker+for+retinal+ganglion+cells.">RNA binding protein with multiple splicing: a new marker for retinal ganglion cells.</a> Invest Ophthalmol Vis Sci. 51 (2), 1052-1058 (2010).</li><li>Van Bergen, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recharacterization+of+the+RGC-5+retinal+ganglion+cell+line.">Recharacterization of the RGC-5 retinal ganglion cell line.</a> Invest Ophthalmol Vis Sci. 50 (9), 4267-4272 (2009).</li><li>Wood, J. P., Chidlow, G., Tran, T., Crowston, J. G., Casson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+differentiation+protocols+for+RGC-5+cells.">A comparison of differentiation protocols for RGC-5 cells.</a> Invest Ophthalmol Vis Sci. 51 (7), 3774-3783 (2010).</li><li>Barres, B. A., Silverstein, B. E., Corey, D. P., Chun, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological,+morphological,+and+electrophysiological+variation+among+retinal+ganglion+cells+purified+by+panning.">Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning.</a> Neuron. 1 (9), 791-803 (1998).</li><li>Hong, S., Iizuka, Y., Kim, C. Y., Seong, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+primary+mouse+retinal+ganglion+cells+using+immunopanning-magnetic+separation.">Isolation of primary mouse retinal ganglion cells using immunopanning-magnetic separation.</a> Mol Vis. 18, 2922-2930 (2012).</li><li>Julius, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sensitivity+of+exponentials+and+other+curves+to+their+parameters.">The sensitivity of exponentials and other curves to their parameters.</a> Comput Biomed Res. 5 (5), 473-478 (1972).</li><li>Reif, A. E., Allen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Akr+Thymic+Antigen+and+Its+Distribution+in+Leukemias+and+Nervous+Tissues.">The Akr Thymic Antigen and Its Distribution in Leukemias and Nervous Tissues.</a> J Exp Med. 120, 413-433 (1964).</li><li>Watanabe, M., Noguchi, T., Tsukada, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional,+cellular,+and+subcellular+distribution+of+Thy-1+antigen+in+rat+nervous+tissues.">Regional, cellular, and subcellular distribution of Thy-1 antigen in rat nervous tissues.</a> Neurochem Res. 6 (5), 507-519 (1981).</li><li>Haeryfar, S. M., Hoskin, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thy-1:+more+than+a+mouse+pan-T+cell+marker.">Thy-1: more than a mouse pan-T cell marker.</a> J Immunol. 173 (6), 3581-3588 (2004).</li><li>Sahagun, G., Moore, S. A., Fabry, Z., Schelper, R. L., Hart, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+murine+endothelial+cell+cultures+by+flow+cytometry+using+fluorescein-labeled+griffonia+simplicifolia+agglutinin.">Purification of murine endothelial cell cultures by flow cytometry using fluorescein-labeled griffonia simplicifolia agglutinin.</a> Am J Pathol. 134 (6), 1227-1232 (1989).</li><li>Shoge, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+retinal+ganglion+cells+culture+enriched+with+the+magnetic+cell+sorter.">Rat retinal ganglion cells culture enriched with the magnetic cell sorter.</a> Neurosci Lett. 259 (2), 111-114 (1999).</li><li>Chintalapudi, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Molecular+Profiling+of+Primary+Mouse+Retinal+Ganglion+Cells:+Comparison+of+Phenotypes+from+Healthy+and+Glaucomatous+Retinas.">Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas.</a> Front Aging Neurosci. 8, 93 (2016).</li><li>Nagdeve, N. G., Yaddanapudi, S., Pandav, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+different+doses+of+ketamine+on+intraocular+pressure+in+anesthetized+children.">The effect of different doses of ketamine on intraocular pressure in anesthetized children.</a> J Pediatr Ophthalmol Strabismus. 43 (4), 219-223 (2006).</li><li>Ding, C., Wang, P., Tian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+general+anesthetics+on+IOP+in+elevated+IOP+mouse+model.">Effect of general anesthetics on IOP in elevated IOP mouse model.</a> Exp Eye Res. 92 (6), 512-520 (2011).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+C(T)+method.">Analyzing real-time PCR data by the comparative C(T) method.</a> Nat Protoc. 3 (6), 1101-1108 (2008).</li><li>Aerts, J., Nys, J., Arckens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+reproducible+and+straightforward+method+to+perform+in+vivo+ocular+enucleation+in+the+mouse+after+eye+opening.">A highly reproducible and straightforward method to perform in vivo ocular enucleation in the mouse after eye opening.</a> J Vis Exp. (92), e51936 (2014).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+AP-2delta+reduces+retinal+ganglion+cell+numbers+and+axonal+projections+to+the+superior+colliculus.">Loss of AP-2delta reduces retinal ganglion cell numbers and axonal projections to the superior colliculus.</a> Mol Brain. 9 (1), 62 (2016).</li><li>Moshiri, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near+complete+loss+of+retinal+ganglion+cells+in+the+math5/brn3b+double+knockout+elicits+severe+reductions+of+other+cell+types+during+retinal+development.">Near complete loss of retinal ganglion cells in the math5/brn3b double knockout elicits severe reductions of other cell types during retinal development.</a> Dev Biol. 316 (2), 214-227 (2008).</li><li>Danias, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+retinal+ganglion+cell+(RGC)+loss+in+aging+DBA/2NNia+glaucomatous+mice:+comparison+with+RGC+loss+in+aging+C57/BL6+mice.">Quantitative analysis of retinal ganglion cell (RGC) loss in aging DBA/2NNia glaucomatous mice: comparison with RGC loss in aging C57/BL6 mice.</a> Invest Ophthalmol Vis Sci. 44 (12), 5151-5162 (2003).</li><li>Jakobs, T. C., Ben, Y., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD15+immunoreactive+amacrine+cells+in+the+mouse+retina.">CD15 immunoreactive amacrine cells in the mouse retina.</a> J Comp Neurol. 465 (3), 361-371 (2003).</li><li>Uusitalo, M., Schlotzer-Schrehardt, U., Kivela, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+localization+of+the+HNK-1+carbohydrate+epitope+to+glial+and+neuronal+cells+of+the+human+retina.">Ultrastructural localization of the HNK-1 carbohydrate epitope to glial and neuronal cells of the human retina.</a> Invest Ophthalmol Vis Sci. 44 (3), 961-964 (2003).</li><li>Jackson, C. J., Garbett, P. K., Nissen, B., Schrieber, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+human+endothelium+to+Ulex+europaeus+I-coated+Dynabeads:+application+to+the+isolation+of+microvascular+endothelium.">Binding of human endothelium to Ulex europaeus I-coated Dynabeads: application to the isolation of microvascular endothelium.</a> J Cell Sci. 96 ( Pt 2), 257-262 (1990).</li><li>Pennartz, S., Perraut, M., Pfrieger, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+retinal+ganglion+cells+from+postnatal+rats+by+magnetic+cell+sorting.">Purification of retinal ganglion cells from postnatal rats by magnetic cell sorting.</a> MACSmore. 12 (2), 16-18 (2010).</li><li>Nadal-Nicolas, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Displaced+retinal+ganglion+cells+in+albino+and+pigmented+rats.">Displaced retinal ganglion cells in albino and pigmented rats.</a> Front Neuroanat. 8, 99 (2014).</li><li>Nadal-Nicolas, F. M., Salinas-Navarro, M., Vidal-Sanz, M., Agudo-Barriuso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+methods+to+trace+retinal+ganglion+cells+with+fluorogold:+from+the+intact+optic+nerve+or+by+stereotactic+injection+into+the+optic+tract.">Two methods to trace retinal ganglion cells with fluorogold: from the intact optic nerve or by stereotactic injection into the optic tract.</a> Exp Eye Res. 131, 12-19 (2015).</li><li>Barnstable, C. J., Drager, U. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thy-1+antigen:+a+ganglion+cell+specific+marker+in+rodent+retina.">Thy-1 antigen: a ganglion cell specific marker in rodent retina.</a> Neuroscience. 11 (4), 847-855 (1984).</li><li>Chiu, K., Lau, W. M., Yeung, S. C., Chang, R. C., So, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrograde+labeling+of+retinal+ganglion+cells+by+application+of+fluoro-gold+on+the+surface+of+superior+colliculus.">Retrograde labeling of retinal ganglion cells by application of fluoro-gold on the surface of superior colliculus.</a> J Vis Exp. (16), (2008).</li></ol>

FACS is the technique of choice to purify cell populations. Other isolation methods include immunopanning, magnetic beads, and complement fixation depletion. The advantage of FACS over these other methodologies is based on the simultaneous identification of cell-surface markers with varying degrees of intensity. The fluorescent intensity of the molecule is proportional to the amount of protein expression. Until now, the isolation of RGCs was based solely on Thy1 (CD90) positivity and CD48 negativity15</...

RGCs are terminally differentiated neurons, and therefore, primary cells are required for experimentation. 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. This is especially important for studies that seek to generate potential therapies to promote RGC function and to minimize their death. The degeneration of RGCs is associated with retinal degenerative diseases, such as glaucoma, diabetic retinopathy, and normal aging. Although the specific cellular mechanisms underlying RGC loss are unclear, a series of risk factors have been identified. Lack of oxygenation at the optic nerve head1,2,3 causes RGC death4 and acts as the disturbance of the homeostasis between the activation of excitatory and inhibitory receptors within individual RGCs5,6. A series of challenges impede progress towards the use of these cells for in-depth studies. First, the number of RGCs present in a murine retina is small. RGCs account for less than 1% of total retinal cells7,8,9. Second, most RGC-specific markers are intracellular proteins10,11,12. Selection based upon these markers leaves the cells non-viable, which precludes downstream functional analyses. Finally, currently available protocols are lengthy and lack standardization13,14. Early RGC isolation protocols were based on immunopanning methods. Barres et al.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. Years later, Hong et al. combined magnetic bead isolation techniques with cell sorting strategies to isolate RGCs with higher purity16. The use of magnetic beads is still used in many scientific applications. Together, magnetic beads and flow cytometry protocols improved the purity of isolated cells. However, these purification systems have not yet been standardized for the isolation of murine RGCs from dissociated retinae.
Flow cytometry is a powerful analytical method that measures the optical and fluorescence characteristics of cell suspensions. Cells are analyzed both quantitatively and qualitatively with a high level of sensitivity, providing a multi-dimensional analysis of the cell population. Cellular discrimination is based upon two main physical properties: cell size or surface area and granularity or internal complexity17. A multi-dimensional analysis can be performed by combining antibodies tagged with fluorochromes that have similar excitation wavelengths and different emissions. Flow cytometry is fast, reproducible, and sensitive. Multitpe lasers permit even greater multi-dimensional analyses of single cells by flow cytometry. Thus, it is an attractive methodology for the study of cytological specimens. Fluorescence activated cell sorting (FACS) uses the multi-dimensional phenotypic differences identified by flow cytometry to sort individual cells into distinct subpopulations.
In the last decade, multiple surface and intracellular proteins have been identified as potential biomarkers for the selection of cells, including neurons. Initial studies that sought to isolate RGCs from rats used Thy1 as a ganglion cell marker. Unfortunately, Thy1, aka CD90, has multiple isoforms in other rodent species18,19,20 and is expressed by multiple retinal cell types19,20, making it a non-specific marker for RGCs. Another surface marker, CD48, is found on monocytic populations in the retina, including macrophages and microglia. Using these two surface markers, a modified RGC signature-Thy1+ and CD48neg cells-was developed15,16,21,22. Unfortunately, these two selection criteria are not sufficient to select for a highly enriched RGC population. To address this unmet need, a flow cytometry protocol was developed23 based on multi-layered positive and negative selection criteria using known cell surface markers to enrich and purify primary murine RGCs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-mouse CD15 PE</td>
			<td>BioLegend</td>
			<td>125606</td>
			<td>Clone MC-480</td>
		</tr>
		<tr>
			<td>Anti-mouse CD48 PE-Cy7</td>
			<td>BioLegend</td>
			<td>103424</td>
			<td>Clone HM48-1</td>
		</tr>
		<tr>
			<td>Anti-mouse CD57</td>
			<td>Sigma Aldrich</td>
			<td>C6680-100TST</td>
			<td>Clone VC1.1</td>
		</tr>
		<tr>
			<td>Anti-mouse CD90.2 AF700</td>
			<td>BioLegend</td>
			<td>105320</td>
			<td>Clone 30-H12</td>
		</tr>
		<tr>
			<td>Brilliant Violet 421 Goat Anti-mouse IgG</td>
			<td>BioLegend</td>
			<td>405317</td>
			<td>Clone Poly4053</td>
		</tr>
		<tr>
			<td>Purified Anti-mouse CD16/32</td>
			<td>BioLegend</td>
			<td>101302</td>
			<td>FcgRII/III block, Clone 93</td>
		</tr>
		<tr>
			<td>Zombie Aqua&#160;</td>
			<td>BioLegend</td>
			<td>423102</td>
			<td>Live cell/ Dead cell discrimination</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30071.03</td>
			<td>U.S. origin</td>
		</tr>
		<tr>
			<td>AbC Total Antibody Compensation Bead Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10497</td>
			<td>Multi-species Ig</td>
		</tr>
		<tr>
			<td>Neurobasal Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21103049</td>
			<td>Add serum to media prior to culture.</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010049</td>
			<td>Saline solution</td>
		</tr>
		<tr>
			<td>Dissection Microscope</td>
			<td>Olympus</td>
			<td>SZ-PT Model</td>
			<td>Stereo Microscope</td>
		</tr>
		<tr>
			<td>Sorvall Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>ST 16R</td>
			<td>All centrifugation performed&#160; at RT</td>
		</tr>
		<tr>
			<td>Base Plate &#8211; Dissection Pan</td>
			<td>Fisher Scientific</td>
			<td>SB15233FIM</td>
			<td>A wax plate can also be used</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Aesculap</td>
			<td>5002-7</td>
			<td>4 &#189; inches</td>
		</tr>
		<tr>
			<td>Iris Scissors, Straight</td>
			<td>Aesculap</td>
			<td>1360</td>
			<td>5 &#189; inches</td>
		</tr>
		<tr>
			<td>Falcon 15 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>352097</td>
			<td>Polypropylene tubes</td>
		</tr>
		<tr>
			<td>Falcon 50 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td>352098</td>
			<td>Polypropylene tubes</td>
		</tr>
		<tr>
			<td>BD FACS Tubes</td>
			<td>Fisher Scientific</td>
			<td>352003</td>
			<td>Polypropylene tubes</td>
		</tr>
		<tr>
			<td>40 mm dishes</td>
			<td>MidSci</td>
			<td>TP93040</td>
			<td>Tissue culture treated</td>
		</tr>
		<tr>
			<td>70 &#956;m nylon strainer</td>
			<td>MidSci</td>
			<td>70ICS</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>40 &#956;m nylon strainer</td>
			<td>MidSci</td>
			<td>40ICS</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>&#160;BD 10 mL syringe</td>
			<td>Fisher Scientific</td>
			<td>301604</td>
			<td>Disposable Syringe without needle</td>
		</tr>
		<tr>
			<td>Pestles</td>
			<td>MidSci</td>
			<td>PEST</td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>Wheaton Vials</td>
			<td>Fisher Scientific</td>
			<td>986734</td>
			<td>No Liner</td>
		</tr>
		<tr>
			<td>BD 30 G needle</td>
			<td>Fisher Scientific</td>
			<td>305128</td>
			<td>1 inch</td>
		</tr>
		<tr>
			<td>Hausser Scientific Bright-Line Glass Counting Chamber</td>
			<td>Fisher Scientific</td>
			<td>0267151B</td>
			<td>Hemocytometer</td>
		</tr>
		<tr>
			<td>Gibco Trypan blue 0.4% Solution</td>
			<td>Fisher Scientific</td>
			<td>15250061</td>
			<td>Viability Dye</td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Fisher Scientific</td>
			<td>05-402-25</td>
			<td>1.5mL</td>
		</tr>
		<tr>
			<td>EVOS Floid Cell Imaging</td>
			<td>Thermo Fisher Scientific</td>
			<td>447113</td>
			<td>Fluorescence Imaging with a 20x objective</td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Fisher Scientific</td>
			<td>04-355-452</td>
			<td>Used to make 70% Ethanol</td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>Rainin</td>
			<td>17014282</td>
			<td>LTS Pipette</td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>Rainin</td>
			<td>17014391</td>
			<td>LTS Pipette</td>
		</tr>
		<tr>
			<td>Pipet-Lite LTS Pipette L-20XLS+</td>
			<td>Rainin</td>
			<td>17014392</td>
			<td>LTS Pipette</td>
		</tr>
		<tr>
			<td>Rack LTS 1000 mL &#8211; GPS-L1000S</td>
			<td>Rainin</td>
			<td>17005088</td>
			<td>Blue Rack Sterile Tips</td>
		</tr>
		<tr>
			<td>Rack LTS 250 mL &#8211; GPS-L250S</td>
			<td>Rainin</td>
			<td>17005092</td>
			<td>Green Rack Sterile Tips</td>
		</tr>
		<tr>
			<td>Rack LTS 20 mL &#8211; GPS-L10S</td>
			<td>Rainin</td>
			<td>17005090</td>
			<td>Red Rack Sterile Tips</td>
		</tr>
		<tr>
			<td>FACSAria II Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td>Custom order</td>
		</tr>
		<tr>
			<td>LSR II Cytometer</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td>Custom order</td>
		</tr>
		<tr>
			<td>Abca8a</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00462440_m1</td>
			<td>M&#252;ller cells</td>
		</tr>
		<tr>
			<td>Aldh1al</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00657317_m1</td>
			<td>M&#252;ller cells</td>
		</tr>
		<tr>
			<td>Aqp4</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00802131_m1</td>
			<td>Astrocytes</td>
		</tr>
		<tr>
			<td>Calb2</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00801461_m1</td>
			<td>Amacrine, Horizontal</td>
		</tr>
		<tr>
			<td>Cd68</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm03047340_m1</td>
			<td>Retinal Pigment Epithelial Cells</td>
		</tr>
		<tr>
			<td>Gad2</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00484623_m1</td>
			<td>Amacrine</td>
		</tr>
		<tr>
			<td>Hprt</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm01545399_m1</td>
			<td>House keeping gene</td>
		</tr>
		<tr>
			<td>Lhx1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm01297482_m1</td>
			<td>Horizontal</td>
		</tr>
		<tr>
			<td>Lim2</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00624623_m1</td>
			<td>Horizontal</td>
		</tr>
		<tr>
			<td>Nrl</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00476550_m1</td>
			<td>Photoreceptors</td>
		</tr>
		<tr>
			<td>Ntrk1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm01219406_m1</td>
			<td>Horizontal</td>
		</tr>
		<tr>
			<td>Pcp4</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00500973_m1</td>
			<td>Bipolar, Amacrine</td>
		</tr>
		<tr>
			<td>Pou4f1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm02343791_m1</td>
			<td>Retinal Ganglion Cells</td>
		</tr>
		<tr>
			<td>Prdx6</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00725435_s1</td>
			<td>Astrocytes</td>
		</tr>
		<tr>
			<td>Prkca</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00440858_m1</td>
			<td>Bipolar</td>
		</tr>
		<tr>
			<td>Prox1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00435969_m1</td>
			<td>Horizontal</td>
		</tr>
		<tr>
			<td>Pvalb</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00443100_m1</td>
			<td>Amacrine</td>
		</tr>
		<tr>
			<td>Rbpms</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm02343791_m1</td>
			<td>Retinal Ganglion Cells</td>
		</tr>
		<tr>
			<td>Rom1</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00436364_g1</td>
			<td>Photoreceptors</td>
		</tr>
		<tr>
			<td>Rpe65</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00504133_m1</td>
			<td>Retinal Pigment Epithelial cells</td>
		</tr>
		<tr>
			<td>Slc1a3</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00600697_m1</td>
			<td>Astrocytes</td>
		</tr>
		<tr>
			<td>Slc6a9</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00433662_m1</td>
			<td>Amacrine</td>
		</tr>
		<tr>
			<td>Sncg</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00488345_m1</td>
			<td>Retinal Ganglion Cells</td>
		</tr>
		<tr>
			<td>Tubb3</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm00727586_s1</td>
			<td>Retinal Ganglion Cells</td>
		</tr>
		<tr>
			<td>Vim</td>
			<td>Thermo Fisher Scientific</td>
			<td>Mm01333430_m1</td>
			<td>M&#252;ller cells</td>
		</tr>
		<tr>
			<td>Taqman Universal Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4440047</td>
			<td>qPCR Reagent</td>
		</tr>
		<tr>
			<td>miRNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>RNA Isolation</td>
		</tr>
		<tr>
			<td>SuperScript VILO cDNA Synthesis Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>11754250</td>
			<td>cDNA synthesis</td>
		</tr>
		<tr>
			<td>Taqman PreAmp Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4391128</td>
			<td>Pre-Amplification step</td>
		</tr>
		<tr>
			<td>BD Cytofix/ Cytoperm</td>
			<td>BD Biosciences</td>
			<td>554714</td>
			<td>Fixation/ Permeabilization Buffer</td>
		</tr>
		<tr>
			<td>BD Perm/ Wash</td>
			<td>BD Biosciences</td>
			<td>554723</td>
			<td>Permeabilization Solution</td>
		</tr>
		<tr>
			<td>RBPMS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-86815</td>
			<td>intracellular antibody</td>
		</tr>
		<tr>
			<td>SNCG</td>
			<td>Gene Tex</td>
			<td>GTX110483</td>
			<td>intracellular antibody</td>
		</tr>
		<tr>
			<td>BRN3A</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-8429</td>
			<td>intracellular antibody</td>
		</tr>
		<tr>
			<td>TUJ1</td>
			<td>BioLegend</td>
			<td>801202</td>
			<td>intracellular antibody</td>
		</tr>
	</tbody>
</table>

isolation of primary murine retinal ganglion cells (rgcs) by flow cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, including diabetic retinopathy and glaucoma, in addition to normal aging. 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. In addition, current isolation methods use intracellular markers to identify RGCs, which produce non-viable cells. These techniques also involve lengthy isolation protocols, so there is a lack of practical, standardized, and dependable methods to obtain and isolate RGCs. 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. 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.

The in-depth study of RGCs is impeded by many factors, including their low frequency and the lack of a robust and standardized methodology for their isolation. Figure 1 shows the methodology used for retinae isolation. Variations in the enucleation procedure exist based on the type of analysis, such as if the enucleation is part of in vivo experimentation27. Enucleation in this protocol is performed on euthanized mice. As show...

Watch this Scientific Journal Video about Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry at JoVE.com

Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

Millions of people suffer from retinal degenerative diseases that result in irreversible blindness. A common element of many of these diseases is the loss of retinal ganglion cells (RGCs). This detailed protocol describes the isolation of primary murine RGCs by positive and negative selection with flow cytometry.

Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry

Neurodegenerative diseases often have a devastating impact on those affected. Retinal ganglion cell (RGC) loss is implicated in an array of diseases, ...

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