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>
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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. 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(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. 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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, ...

The overall goal of this procedure is to isolate an enriched homogeneous population of primary murine retinal ganglion cells. This method can help answer questions in the retinal ganglion cell field about the mechanisms underlying the declining visual acuity in age populations and in retinal degenerative disease populations. The main advantage of this technique is that it can offer a fast, visible, reproducible, and standardized protocol for the isolation of enriched primary murine retinal ganglion cells.We first had the idea for this method when Sumana Chintalapudi, who was then a graduate student in my lab and first author on this study, presented at our journal club a 2013 paper by Krishna Murtheadol, and their study clearly demonstrated that the RGC five cell line, which was originally generated as an immortalized rat retinal ganglion cell line, was no longer of rat origin, nor did it possess the RGC phenotype. It became apparent to us that this problem left a large gap in the resources available to the vision research community, including our own lab. Sumana and I reached out Dr.Morales-Tirado and together the three of us developed a new method for isolating live and highly enriched RGCs.Generally, individuals new to this method will struggle because the goals of this method are to isolate a live and highly enriched cell population that can be either cultured as primary cells, or immortalized as a cell line. Because of this one must be very careful not to damage the cells that one is trying to isolate. In addition, the parameters of cell sorting method must be tailored for the specific cells one is trying to isolate.Both methods require special skill sets. Demonstrating the procedures will be Dr.Xiang Di Wang, a research associate from Dr.Jablonski's lab, and Mr.Zach Goldsmith, PhD candidate from my laboratory. To enucleate the eye, first insert forceps under the globe of the eye.Grip the optic nerve and pull up. The globe will be enucleated with the optic nerve intact. Place the eye in a vial of PBS on ice, and enucleate the other eye.When all of the eyes have been collected, place one eye in a Petri dish of fresh PBS under a dissection microscope, and use forceps to carefully grasp the globe at the base of the optic nerve. Using a sharp 30 gauge needle, puncture the cornea to allow the aqueous humor to drain making it easier to hold the eye with the forceps. Holding the cornea with the forceps, use scissors to make a small incision in the corneal tissue and use the forceps to gently peel away the cornea and the sclera.When the globe is peeled halfway, use the forceps to roll out the retina and the lens. Place the retina in a small 40 millimeter Petri dish containing PBS and 1%FPS in a biosafety cabinet and wash the retina three times in fresh PBS plus FBS for each wash. When all of the eyes have been washed, place up to 12 retinae onto a sterile 70 micron nylon strainer moistened with PBS and FBS and gently macerate the retinae with the back end of a 10 milliliter syringe plunger, using a circular motion.When all of the cells have been dissociated place the strainer over a polypropylene collection tube and use a P1000 pipette to filter the cells through the strainer into the collection tube. Rinse the strainer with PBS and FBS, pooling the wash in the collection tube. Add enough PBS plus FBS to bring the final volume up to one milliliter of solution per retina and collect the cells by centrifugation.Then, resuspend the pellet in PBS plus FBS at a one milliliter of medium per five retinae concentration, if immunolabeling is to be performed immediately. Alternatively, incubate the cells resuspended in neural cell medium overnight at four degrees Celsius in the horizontal position. To immunolabel the retinal cells, centrifuge the retinal cell suspension, and resuspend the pellet in 50 microliters of fresh PBS plus FBS in a fax tube.Set aside five times 10 to the sixth cells for the unlabeled negative control. Next, block any non specific FC receptor binding in each tube with one microliter of anti mouse CD 16 32 antibody per one times 10 to the sixth cells, for 10 minutes at room temperature. At the end of the blocking incubation, add the antibody cocktail of interest to the cells with gentle pipetting for a 30 minute incubation on ice protected from light.Then wash the cells two times in a formula liter final volume of PBS and FBS. Label the cells with an appropriate secondary antibody for another 30 minutes on ice protected from light, followed by two washes in PBS plus FBS as just demonstrated. Resuspend the pellets in fresh PBS and FBS for counting cells.To adjust the compensation add three drops of polystyrene microspheres into one sterile fax tube per florafor and one microgram of the respective florafor to each tube. Incubate the microspheres for 15 minutes at room temperature protected from light. Then wash the microspheres in three milliliters of PBS and FBS and carefully remove the supernatant.Resuspend the microsphere pellets in 250 microliters of PBS and FBS and load the unlabeled sample tube onto the flow cytometer. In the flow cytometer software, select experiment, compensation set up, and create compensation controls. Add the florafor specific controls from the displayed list and click OK.Verify the forward scattered and side scattered light, and gate the initial population.Then install the negative control tube onto the cytometer and click load. Verify that the population of interest or P1 is displayed and select the population. Right click the P1 gate and select calculate compensation.Then click record data. When the recording is finished, click unload to remove the tube and load the next control tube to adjust the compensation controls. When all of the control samples have been recorded, create a forward versus side scatter plot for the first experimental tube, and gate the P1 population.Next, open a side scatter height versus side scatter width pseudo color plot and gate the single cells. Using the single cells, create a forward scatter height versus forward scatter width plot, and gate to exclude the dublets. Then generate a CD48 versus CD90.2 pseudo color plot and gate the CD90.2 positive CD48 negative cells to eliminate the monocytes, followed by a CD57 versus CD15 pseudo color plot to eliminate any amacrine cell contaminants.Now define the populations to be collected for the sample in the sort layout sheet, and sort the cells. At the end of the sort, use a 2.5 times 10 to the fourth cell aliquot to confirm the purity of the isolated cell population. After meshing the retinae in the cell strainer multiple inner retinal cells can be visualized, even though the retinal ganglion cells have lost their signature morphology, due to axotomy during the cell isolation procedure.The classic dye one positive CD48 negative surface phenotype is not sufficient for identifying and isolating the murine retinal ganglion cells, as these cells express genes associated with amacrine, muller, bipolar, horizontal photo receptor, and retinal pigment epithelial cells. The sorted cells also express retinal ganglion cell associated intracellular markers such as synuclein gamma, BRN3A, TUJ1, and RBPMS, with the intracellular localization of the markers further confirmed by imaging flow cytometry. Some of the sorted cells even begin to exhibit retinal ganglion cell morphology after in vitro cell culture.Further, quantitative PCR analysis of the highly enriched sorted cell population reveals a many fold increase in the genes coding for retinal ganglion specific intracellular markers. Once mastered, this technique can be completed in less than six hours if it's performed properly. While attempting this procedure, it's important to remember to keep your cell suspension sterile, to block the FC receptors from the cell suspension, to avoid non specific binding of the antibodies, and to discuss your cell sorting details or strategy with the cell sorter operator.Following this procedure, other methods like QPCR can be performed to answer additional questions about gene expression profiling. Don't forget that working specialized equipment, such as a cell sorter, requires a highly specialized operator. After its development, this technique paved the way for researchers in the vision field to help understand survival and death mechanisms in retinal ganglion cells, for the development of novel therapeutics for vision preservation.After watching this video, you should have a good understanding of how to isolate an enriched homogeneous population of primary murine retinal ganglion cells.

Research

JoVE Journal

Bioengineering

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