作者要感谢微生物学，免疫学和生物化学系高级插图师Tim Higgins先生提供技术视频协助; Matthew W. Wilson博士进行讨论，还有Jablonski和Morales-Tirado实验室的成员对他们的帮助意见。这项工作得到了阿尔康研究院青年研究员奖（VMM-T），田纳西大学研究基金会（VMM-T），国家眼科学院EY021200（MMJ），格温奖学金（VMM-T）等的支持。格温博士后研究金（ZKG），国防军医学研究和物资司令部（VMM-T）以及研究预防盲人无限制赠款。

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作者宣称他们没有竞争的经济利益。

FACS是净化细胞群体的首选技术。其他分离方法包括免疫沉淀，磁珠和补体固定消耗。 FACS与这些其他方法的优点是基于同时鉴别具有不同程度的强度的细胞表面标志物。分子的荧光强度与蛋白质表达量成比例。到目前为止，RGC的分离仅基于Thy1（CD90）阳性和CD48阴性15,16,22,34，而不管使用的分离方法如何。最近已经显示，Thy1 + CD48 neg表型不足以分离表达RGC细胞内标志物...

RGC是终末分化的神经元，因此，原代细胞是实验所必需的。用于分离和富集原代小鼠视网膜神经节细胞（RGC）的方案的开发是揭示RGC健康和体外变性的机制的基础。这对于寻求产生潜在疗法以促进RGC功能并尽量减少死亡率的研究尤为重要。 RGC的变性与视网膜退行性疾病如青光眼，糖尿病性视网膜病变和正常衰老有关。虽然RGC损失的具体细胞机制尚不清楚，但已经确定了一系列危险因素。缺乏视神经头1,2,3的氧合导致RGC死亡4 ，并且作为兴奋性和i激活之间的动态平衡的干扰个体RGCs内的抑制性受体5,6 。一系列挑战妨碍了使用这些细胞进行深入研究的进展。首先，鼠视网膜中RGCs的数量很少。 RGC占总视网膜细胞7,8,9的不到1％。第二，大多数RGC特异性标记物是细胞内蛋白质10,11,12 。基于这些标记的选择使细胞不可行，这排除了下游功能分析。最后，目前可用的协议冗长，缺乏标准化13,14 。早期RGC分离方案是基于免疫方法。 Barres 等人 15 适应经典免疫基于对抗胸腺细胞抗原（也称Thy1）（细胞表面标记）的免疫阳性，在阳性选择之前，将大部分视网膜细胞从单核细胞和内皮细胞排除第二步。几年后，Hong et al。组合磁珠分离技术与细胞分选策略分离更高纯度的RGC 16 。在许多科学应用中仍然使用磁珠。一起，磁珠和流式细胞术方案提高了分离细胞的纯度。然而，这些纯化系统尚未被标准化用于从解离的视网膜分离鼠RGCs。 流式细胞仪是一种强大的分析方法，可以测量细胞悬浮液的光学和荧光特性。以高水平的灵敏度定量和定性分析细胞，为细胞群体提供多维分析通货膨胀。细胞鉴别基于两个主要物理性质:细胞大小或表面积，粒度或内部复杂性17 。可以通过组合具有类似激发波长和不同排放物的荧光染料标记的抗体来进行多维分析。流式细胞术是快速，可重复和敏感的。多重激光器通过流式细胞术允许单个细胞的更多的多维分析。因此，它是研究细胞学标本的有吸引力的方法。荧光激活细胞分选（FACS）使用通过流式细胞术鉴定的多维表型差异将单个细胞分类为不同的亚群。 在过去十年中，多种表面和细胞内蛋白已被鉴定为用于选择细胞（包括神经元）的潜在生物标志物。试图从大鼠中分离RGC的初步研究使用Thy1作为神经节细胞l标记。不幸的是，Thy1， 也就是 CD90，在其他啮齿动物物种18,19,20中具有多个同种型，并且由多个视网膜细胞类型19,20表达，使其成为RGC的非特异性标记。在视网膜中的单核细胞群体中发现另一个表面标志物CD48，包括巨噬细胞和小神经胶质细胞。使用这两个表面标记，修饰的RGC特征-Thy1 +和CD48 neg细胞 - 被开发出15,16,21,22。不幸的是，这两个选择标准不足以选择高度富集的研资局人口。为了解决这一未满足的需求，基于多层次正，负选择标准，开发了流式细胞仪方案23已知的细胞表面标志物以富集和纯化原代鼠RGC。

<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

神经退行性疾病往往对受影响者有破坏性的影响。除了正常老化之外，视网膜神经节细胞（RGC）损失也涉及一系列疾病，包括糖尿病性视网膜病变和青光眼。尽管它们的重要性，RGC迄今为止非常难以研究，部分原因在于它们仅占视网膜中各种细胞的一小部分。此外，目前的分离方法使用细胞内标记来鉴定产生非活细胞的RGC。这些技术还涉及冗长的分离方案，因此缺乏获得和分离RGC的实用，标准化和可靠的方法。这项工作描述了一种有效，全面和可靠的方法，使用基于正选择标准和阴性选择标准的方案从小鼠视网膜分离原代RGC。所提出的方法允许未来RGCs的研究，目的是更好地了解专业由神经退行性疾病中功能性RGCs的丧失导致的视力下降。

<html> RGC的深入研究受到许多因素的阻碍，包括其频率低，缺乏强大和标准化的隔离方法。 图1显示了用于视网膜分离的方法。根据分析方法存在摘除程序的变化，例如摘除核素是体内实验的一部分27 。在安乐死小鼠中进行该方案的摘除。 如图1A- B所示，镊子被放置在眼睛下方并被拉起以引?...

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

数百万人患有导致不可逆失明的视网膜退行性疾病。许多这些疾病的常见因素是视网膜神经节细胞（RGC）的丧失。该详细方案描述了通过流式细胞术的阳性和阴性选择分离原代鼠RGC。

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

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

14.9K 次观看.  University of Tennessee Health Science Center. 数百万人患有导致不可逆失明的视网膜退行性疾病。许多这些疾病的常见因素是视网膜神经节细胞（RGC）的丧失。该详细方案描述了通过流式细胞术的阳性和阴性选择分离原代鼠RGC。 

视频: 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.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

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

科研

神经科学

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.

小鼠视网膜内皮细胞分离用于二代测序

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

发育生物学

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.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

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

免疫与感染

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.

视网膜神经节细胞定义子集的单细胞RNA-Seq

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.

两种剥离方法，用于分离小鼠视网膜中的光感受器细胞区室以进行蛋白质分析

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

培养的原代鼠 RPE 的表征和形态动力学

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.

作者聚焦：从成年小鼠中有效分离原代视网膜色素上皮细胞用于视网膜病变研究

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.

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

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

<meta charset="utf8"></head><body>小鼠全支架视网膜中 RGC 的免疫染色和基于 AI 的计数

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.

<meta charset="utf8"></head><body>作者聚焦:青光眼小鼠模型中的高效视网膜神经节细胞计数用于治疗评估

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.

小鼠视网膜神经节细胞的全支架免疫染色和自动计数

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

通过流式细胞术分离原代小鼠视网膜神经节细胞（RGC）

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此视频中的章节

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

小鼠视网膜内皮细胞分离用于二代测序

评估视网膜小胶质细胞吞噬功能

视网膜神经节细胞定义子集的单细胞RNA-Seq

两种剥离方法，用于分离小鼠视网膜中的光感受器细胞区室以进行蛋白质分析

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

一种从成年小鼠中分离和培养视网膜色素上皮细胞的简化方法

小鼠视网膜神经节细胞的全支架免疫染色和自动计数

小鼠视网膜神经节细胞的全支架免疫染色和自动计数