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.

<ol>
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The authors declare that they have no competing financial interests.

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><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&amp;nbsp;</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&amp;nbsp; at RT</td></tr><tr><td>Base Plate &amp;ndash; 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 &amp;frac12; inches</td></tr><tr><td>Iris Scissors, Straight</td><td>Aesculap</td><td>1360</td><td>5 &amp;frac12; 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 &amp;mu;m nylon strainer</td><td>MidSci</td><td>70ICS</td><td>sterile</td></tr><tr><td>40 &amp;mu;m nylon strainer</td><td>MidSci</td><td>40ICS</td><td>sterile</td></tr><tr><td>&amp;nbsp;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 &amp;ndash; GPS-L1000S</td><td>Rainin</td><td>17005088</td><td>Blue Rack Sterile Tips</td></tr><tr><td>Rack LTS 250 mL &amp;ndash; GPS-L250S</td><td>Rainin</td><td>17005092</td><td>Green Rack Sterile Tips</td></tr><tr><td>Rack LTS 20 mL &amp;ndash; 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>&lt;em&gt;Abca8a&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00462440_m1</td><td>M&amp;uuml;ller cells</td></tr><tr><td>&lt;em&gt;Aldh1al&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00657317_m1</td><td>M&amp;uuml;ller cells</td></tr><tr><td>&lt;em&gt;Aqp4&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00802131_m1</td><td>Astrocytes</td></tr><tr><td>&lt;em&gt;Calb2&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00801461_m1</td><td>Amacrine, Horizontal</td></tr><tr><td>&lt;em&gt;Cd68&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm03047340_m1</td><td>Retinal Pigment Epithelial Cells</td></tr><tr><td>&lt;em&gt;Gad2&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00484623_m1</td><td>Amacrine</td></tr><tr><td>&lt;em&gt;Hprt&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm01545399_m1</td><td>House keeping gene</td></tr><tr><td>&lt;em&gt;Lhx1&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm01297482_m1</td><td>Horizontal</td></tr><tr><td>&lt;em&gt;Lim2&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00624623_m1</td><td>Horizontal</td></tr><tr><td>&lt;em&gt;Nrl&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00476550_m1</td><td>Photoreceptors</td></tr><tr><td>&lt;em&gt;Ntrk1&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm01219406_m1</td><td>Horizontal</td></tr><tr><td>&lt;em&gt;Pcp4&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00500973_m1</td><td>Bipolar, Amacrine</td></tr><tr><td>&lt;em&gt;Pou4f1&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm02343791_m1</td><td>Retinal Ganglion Cells</td></tr><tr><td>&lt;em&gt;Prdx6&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00725435_s1</td><td>Astrocytes</td></tr><tr><td>&lt;em&gt;Prkca&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00440858_m1</td><td>Bipolar</td></tr><tr><td>&lt;em&gt;Prox1&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00435969_m1</td><td>Horizontal</td></tr><tr><td>&lt;em&gt;Pvalb&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00443100_m1</td><td>Amacrine</td></tr><tr><td>&lt;em&gt;Rbpms&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm02343791_m1</td><td>Retinal Ganglion Cells</td></tr><tr><td>&lt;em&gt;Rom1&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00436364_g1</td><td>Photoreceptors</td></tr><tr><td>&lt;em&gt;Rpe65&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00504133_m1</td><td>Retinal Pigment Epithelial cells</td></tr><tr><td>&lt;em&gt;Slc1a3&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00600697_m1</td><td>Astrocytes</td></tr><tr><td>&lt;em&gt;Slc6a9&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00433662_m1</td><td>Amacrine</td></tr><tr><td>&lt;em&gt;Sncg&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00488345_m1</td><td>Retinal Ganglion Cells</td></tr><tr><td>&lt;em&gt;Tubb3&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm00727586_s1</td><td>Retinal Ganglion Cells</td></tr><tr><td>&lt;em&gt;Vim&lt;/em&gt;</td><td>Thermo Fisher Scientific</td><td>Mm01333430_m1</td><td>M&amp;uuml;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, ...

isolation-primary-murine-retinal-ganglion-cells-rgcs-flow

Research

JoVE Journal

Bioengineering

15.0K Views.  University of Tennessee Health Science Center.  Miljoenen mensen lijden aan retinale degeneratieve ziekten die leiden tot onomkeerbare blindheid. Een gemeenschappelijk element van veel van deze ziekten is het verlies van retinale ganglioncellen (RGC's). Dit gedetailleerde protocol beschrijft de isolatie van primaire murine RGC's door positieve en negatieve selectie met flowcytometrie. 

Video: Isolation of Primary Murine Retinal Ganglion Cells RGCs by Flow Cytometry

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Neuroscience

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies ...

Developmental Biology

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Immunology and Infection

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Immunostaining and AI-Based Counting of RGCs in Mouse Whole-Mount Retina

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve as valuable animal models for studying the pathogenesis and treatment of glaucoma due to their relatively homogenous genetic background and retinal ganglion cells (RGCs), which structurally resemble those in humans. Accurate assessment of RGC damage and treatment outcomes in mouse glaucoma models requires determining the RGC number across the entire retina. This protocol outlines a comprehensive method involving the isolation of the whole retina, labeling RGCs with specific antibodies, and rapid, accurate automatic counting of RGCs using an AI-based program. The streamlined approach allows for efficient and precise quantification of RGC numbers in mouse retinas, facilitating the evaluation of RGC degeneration and potential therapeutic interventions. By enabling researchers to assess the extent of RGC damage, this protocol contributes to a deeper understanding of glaucoma pathogenesis and aids in developing effective treatment strategies to manage and prevent vision loss.

Author Spotlight: Efficient Retinal Ganglion Cell Counting in Mouse Models of Glaucoma for Treatment Evaluation

This protocol describes the procedure for isolating the whole-mount mouse retina and performing immunostaining to label all retinal ganglion cells (RGCs). The process is followed by imaging and automatically counting RGCs using AI-based software, providing a simple, fast, and accurate method for quantifying RGCs in the entire mouse retina.

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Biology

Isolation of Primary Mouse Retinal Glial M&#252;ller Cells

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both the retinal blood vessels and the retinal neurons. Because of their growth, M&#252;ller cells perform several crucial tasks in a healthy retina, including the uptake and recycling of neurotransmitters, retinoic acid compounds, and ions (like potassium K+). In addition to regulating blood flow and maintaining the blood-retinal barrier, they also regulate the metabolism and the supply of nutrients to the retina. An established procedure for isolating primary mouse M&#252;ller cells is presented in this manuscript. To better understand the underlying molecular processes involved in the various mouse models of ocular disorders, M&#252;ller cell isolation is an excellent approach. This manuscript outlines a detailed procedure for M&#252;ller cell isolation from mice. From enucleation to seeding, the entire process lasts about a few hours. For 5-7 days after seeding, the media shouldn&#39;t be changed in order to allow the isolated cells to grow unhindered. Cell characterization using morphology and distinct immunofluorescent markers comes next in the process. Maximum passages for cells are 3-4 times.

This manuscript describes a detailed protocol for isolating retinal glial M&#252;ller cells from mouse eyes. The protocol starts with enucleation and dissection of mouse eyes, followed by isolation, seeding, and culturing of M&#252;ller cells.

The primary supporting cell of the retina is the retinal glial M&#252;ller cell. They cover the entire retinal surface and are in close proximity to both ...

Identification of Mouse Retinal Ganglion Cell Subtypes Through Fluorescent Tracing

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/55229/single-cell-rna-seq-of-defined-subsets-of-retinal-ganglion-cells'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Laboissonniere,&#160;et. al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2017)</a>. &#160;This video demonstrates a fluorescent tracing technique to identify retinal ganglion cell (RGC) subtypes in a transgenic mouse retina. The process involves securing the isolated retina in a recording chamber, maintaining cell viability with oxygenated perfusion, and targeting fluorescently labeled RGCs using a pipette with a tracer. By analyzing dendritic morphology and stratification, the method enables classification of distinct RGC subtypes.

Source:&#160;Laboissonniere,&#160;et. al. Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells.&#160;J. Vis. Exp. (2017).&#160;This video ...

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

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