著者はテクニカルビデオアシスタンスのための免疫学および生化学の微生物学部門のシニアイラストレーター、ティムヒギンズ氏に感謝したい。 Matthew W. Wilson博士とJablonskiとMorales-Tiradoラボのメンバーの皆さんにお話を伺いました。この研究は、アルコン研究所の若手研究者賞（VMM-T）、テネシー大学研究財団（VMM-T）、国立眼科研究所EY021200（MMJ）、ゲーウィンフェローシップ（VMM-T） Gerwin Pre-doctoral Fellowship（ZKG）、国防総省陸軍医学研究院（VMM-T）、および無防備失調症研究のための無制限助成金などが含まれます。

<ol>
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著者らは競合する金銭的利益がないと宣言している。

FACSは、細胞集団を精製するために選択される技術である。他の単離方法には、免疫パンニング、磁気ビーズ、および補体固定枯渇が含まれる。これらの他の方法に対するFACSの利点は、様々な程度の強度を有する細胞表面マーカーの同時同定に基づく。分子の蛍光強度はタンパク質発現量に比例する。今まで、RGCの単離は、使用した単離方法にかかわらず、 Thy1（CD90）陽性およびCD48?...

RGCは最終的に分化したニューロンであるため、実験には一次細胞が必要である。原発性ネズミ網膜神経節細胞（RGC）の単離および濃縮のためのプロトコールの開発は、 インビトロでのRGCの健康および変性の機序を明らかにする上で基本的なものである。これは、RGC機能を促進し、その死を最小限に抑えるための潜在的な治療法を生み出そうとする研究にとって特に重要です。 RGCの変性は、緑内障、糖尿病性網膜症および正常な老化などの網膜変性疾患に関連する。 RGC喪失の根底にある特定の細胞機構は不明であるが、一連の危険因子が同定されている。視神経乳頭1、2、3での酸素供給の欠如は、 RGC死を引き起こし、興奮性物質とiの活性化との間のホメオスタシスの障害として作用する個々のRGC内の阻害性受容体5,6 。一連の課題は、詳細な研究のためにこれらの細胞の使用への進展を妨げる。第1に、ネズミ網膜に存在するRGCの数は少ない。 RGCは全網膜細胞の7 ％ 、 8,9 ％未満を占める。第二に、ほとんどのRGC特異的マーカーは細胞内タンパク質10,11,12である。これらのマーカーに基づく選択は、細胞を生存不能にし、下流の機能的分析を排除する。最後に、現在利用可能なプロトコルは長く、標準化が不十分である 13,14 。初期のRGC単離プロトコールはイムノパニング法に基づいていた。 Barres et al。 15 古典的なイムノープを適応させた細胞表面マーカーである抗胸腺細胞抗原（別名Thy1）に対する免疫陽性に基づくポジティブ選択の前に網膜細胞の大部分から単球および内皮細胞を除外した第2段階を加えた。数年後、香港らは、純度の高いRGCを分離するためのセルソーティング戦略と組み合わせた磁気ビーズ分離技術16磁気ビーズの使用は、多くの科学的用途において依然として使用されている。一緒に、磁気ビーズおよびフローサイトメトリープロトコルは、単離された細胞の純度を改善した。しかし、これらの精製システムは、解離した網膜からのネズミRGCの単離のためにまだ標準化されていない。 フローサイトメトリーは、細胞懸濁液の光学特性および蛍光特性を測定する強力な分析方法です。細胞を高レベルの感度で定量的および定性的に分析して、細胞集団の多次元分析を提供するation。細胞の識別は、細胞の大きさまたは表面積、粒状性または内部の複雑さの2つの主要な物理的特性に基づいています。類似の励起波長および異なる放出を有する蛍光色素で標識された抗体を組み合わせることによって、多次元分析を行うことができる。フローサイトメトリーは、迅速で、再現性があり、感度が高い。マルチタイプレーザーは、フローサイトメトリーによる単一細胞のより大きな多次元分析を可能にする。したがって、それは細胞学的標本の研究のための魅力的な方法論である。蛍光活性化細胞選別（FACS）は、フローサイトメトリーによって同定された多次元表現型の差異を用いて、個々の細胞を異なる亜集団に分類する。 過去10年間に、複数の表面タンパク質および細胞内タンパク質が、ニューロンを含む細胞の選択のための潜在的なバイオマーカーとして同定されている。ラットからRGCを単離することを試みた最初の研究は、Th1を神経節細胞として使用したlマーカー。残念ながら、Thy1、 別名 CD90は、他のげっ歯類種18,19,20に複数のアイソフォームを有し、RGCの非特異的マーカーとなる複数の網膜細胞タイプ19,20によって発現される。別の表面マーカー、CD48は、マクロファージおよびミクログリアを含む網膜の単球集団に見出される。これらの2つの表面マーカーを用いて、改変RGCシグネチャー-Thy1 +およびCD48ネガティブ細胞が開発された（15,16,21,22）。残念なことに、これらの2つの選択基準は、高度に濃縮されたRGC集団を選択するのに十分ではない。この満たされていないニーズに対処するために、フローサイトメトリープロトコルが、多層陽性選択基準および陰性選択基準に基づいて開発された一次ネズミRGCを富化および精製するための既知の細胞表面マーカーを提供する。

<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

神経変性疾患は、しばしば影響を受ける人々に壊滅的な影響を与える。網膜神経節細胞（RGC）の喪失は、正常な老化に加えて、糖尿病性網膜症および緑内障を含む一連の疾患に関係している。彼らの重要性にもかかわらず、RGCは網膜内の多種多様な細胞のわずかな割合しか占めないという事実のために今まで研究することは極めて困難であった。さらに、現在の単離方法は、細胞内マーカーを使用して、非生存細胞を産生するRGCを同定する。これらの手法には長時間の分離プロトコルも含まれるため、RGCを取得して分離するための実用的で標準化された信頼できる方法が欠けています。この研究は、ポジティブおよびネガティブ選択基準に基づくプロトコールを用いて、マウス網膜から一次RGCを単離するための、効率的で包括的で信頼できる方法を記載する。提示された方法は、RGCの将来の研究を可能にし、主要な神経変性疾患における機能性RGCの喪失に起因する視力の低下。

<html> RGCの徹底的な研究は、頻度が低く、分離のための堅牢で標準化された方法論の欠如を含む多くの要因によって妨げられています。 図1は、網膜の隔離のために使用される方法論を示す。除核処置のバリエーションは、除核が生体内実験の一部である場合など、分析のタイプに基づいて存在する（ 27） 。安楽死させた...

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

何百万人もの人々が不可逆的な失明をもたらす網膜変性疾患に苦しんでいます。これらの疾患の多くの共通要素は、網膜神経節細胞（RGC）の喪失である。この詳細なプロトコールは、フローサイトメトリーによる陽性および陰性選択による原発性ネズミRGCの単離を記載する。

フローサイトメトリーによる一次マウス網膜神経節細胞（RGCs）の単離

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 ビュー.  University of Tennessee Health Science Center. 何百万人もの人々が不可逆的な失明をもたらす網膜変性疾患に苦しんでいます。これらの疾患の多くの共通要素は、網膜神経節細胞（RGC）の喪失である。この詳細なプロトコールは、フローサイトメトリーによる陽性および陰性選択による原発性ネズミRGCの単離を記載する。 

ビデオ: 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.

遺伝的に標識された網膜神経節細胞からの光は応答を記録するために隔離された網膜の準備

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

神経科学

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.

次世代シーケンシングのためのマウス網膜内皮細胞の単離

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

発生生物学

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.

網膜ミクログリアの貪食機能を評価します<em&gt;インビボ</em&gt;フローサイトメトリーベースのアッセイを使用しました

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・感染学

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.

網膜神経節細胞の定義されたサブセットの単一細胞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.

タンパク質分析のためのマウス網膜における光受容体細胞区画の単離のための2つの剥離方法

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

培養初代マウスRPEの特性評価と形態学的ダイナミクス

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.

著者スポットライト:網膜症研究のための成体マウスからの一次網膜色素上皮細胞の効率的な単離

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.

成体マウスからの網膜色素上皮細胞の単離および培養のための簡易法

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>マウスホールマウント網膜におけるRGCの免疫染色とAIベースのカウント

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>著者スポットライト:治療評価のための緑内障マウスモデルにおける効率的な網膜神経節細胞カウント

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.

マウス網膜神経節細胞のホールマウント免疫染色および自動計数(英語)

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

フローサイトメトリーによる一次マウス網膜神経節細胞（RGCs）の単離

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遺伝的に標識された網膜神経節細胞からの光は応答を記録するために隔離された網膜の準備

次世代シーケンシングのためのマウス網膜内皮細胞の単離

網膜ミクログリアの貪食機能を評価します

網膜神経節細胞の定義されたサブセットの単一細胞RNA-Seq

タンパク質分析のためのマウス網膜における光受容体細胞区画の単離のための2つの剥離方法

成体マウスからの網膜色素上皮細胞の単離および培養のための簡易法

成体マウスからの網膜色素上皮細胞の単離および培養のための簡易法

成体マウスからの網膜色素上皮細胞の単離および培養のための簡易法

マウス網膜神経節細胞のホールマウント免疫染色および自動計数(英語)

マウス網膜神経節細胞のホールマウント免疫染色および自動計数(英語)