Name: assessing retinal microglial phagocytic function in vivo using a flow cytometry-based assay
Uploaded: 2016-10-18T15:00:00
Description: Watch this Scientific Journal Video about Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay at JoVE.com

Salome Murinello is supported by American Diabetes Association grant #1-16-PDF-072. This work was supported by grants to Martin Friedlander from the National Institutes of Health (National Eye Institute EY11254 and EY22025) and the Lowy Medical Research Institute.

全ての動物は、スクリップス研究所によって確立された倫理ガイドラインに従って処理しました。 注射用材料の調製<ol><li> 33 G針と注射器を滅菌：C.ο115で分解し、オートクレーブ滅菌リン酸緩衝生理食塩水（PBS）に上昇することによって、注射用の針を準備します。 </li><li> 10分 - 5室温で蛍光標識された粒子を解凍します。 Ca 2+ / Mgの2+との無菌PBS中に50mg / mlの...

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この方法では3つの重要なステップがあります：蛍光標識された粒子の（1）硝子体内注射; （2）収穫および網膜組織の調製。 （3）フローサイトメトリー分析。私たちは、研究者は、我々がここで紹介する方法を実行する前に硝子体内注射を練習することをお勧めします。アルビノマウス（ 例えば 、BALB / c）および着色した溶液（ 例えば、蛍光標識された粒子）が針と注入した?...

この方法の全体的な目標を正確に評価し、 生体内ミクログリア食作用に定量化することです。ミクログリアは、中枢神経系（CNS）の組織常在性マクロファージです。彼らは、組織恒常性の維持を確実にするために様々な機能を実行します。これらは、免疫監視機構、神経栄養因子の分泌と、極めて重要で、貪食1が含まれます。ミクログリアの貪食は、無関係なシナプス（シナプス剪定）の食作用およびアポトーシスニューロン2-4の除去などの脳や網膜の開発中に、いくつかの重要なイベント、内のキーです。また、損傷を受けたまたはアポトーシスニューロンのミクログリア食作用、細胞の破片、および侵入微生物が成人期5を介して中枢神経系の恒常性を維持するために必須であることが示されています。最後に、ミクログリア食作用は、アルツハイマー病、年齢関連を含むいくつかの神経変性疾患の病因に関与しています不良または不十分な貪食能は、アミロイドβ（Aβ）斑とドルーゼン、それぞれ6,7のビルドアップに寄与し得ることが示唆されている黄斑変性症、。 ミクログリア機能はしっかり特に、腫瘍増殖因子β、または細胞 - 細胞相互作用などの可溶性因子によって、それらの微小環境によって調節されます。ミクログリアは、排他的にそれぞれの受容体CD200RとCX3CR1を表現しながら、ニューロンは恒常的に、このようなCD200およびCX3CL1などのいくつかの細胞表面リガンドを発現します。これらの受容体は、それらの細胞内部分に、免疫レセプターチロシンベース阻害モチーフ（のITIM）を含有します。これらの受容体は、神経炎症に寄与することができる、ミクログリアの過剰刺激を防止するために重要である阻害剤。したがって、通常の生理学的条件下で、ニューロンとミクログリアの間の細胞間相互作用は、休止状態にミクログリアを保ちます。組織損傷の間、ただし、ニューロンは、EXPをダウンレギュレートすることができこれらのリガンドのression、ミクログリアの活性化に対する阻害効果を除去します。 （食作用を含む）ミクログリアの機能は、このようにしっかりとその微小環境8に連結されています。それにもかかわらず、現在までに、生理的な文脈で、または完全にそのCNS微小環境を複製する方法で、ミクログリアの貪食作用を研究するための標準化アッセイはありません。 いくつかのアッセイは、初代ミクログリアまたはミクログリア細胞株は、標的細胞（ 例えば 、アポトーシスのニューロン）または蛍光標識されたビーズで培養し、インビトロでミクログリアの食作用活性を測定するために開発されてきました。標的取り込みはその後、蛍光イメージング顕微鏡を用いて評価またはサイトメトリー9-12フローです。これらのアッセイは、薬理学的または遺伝的操作が有益ながら、完全に生体内環境での複合体を複製するために失敗し、ミクログリア食作用に影響を与えることができるかのテストを可能にします。ミクログリア食作用を調べるための間接的な方法in vivoで報告されている。これらは、食作用に関与すると考えられ、分子の染色によって達成されている（ 例えば、CD68）、（ 例えば 、妥協ニューロンまたはシナプス要素）食作用のためにミクログリアとターゲットの物理的な近接性を評価する、または食作用の免疫組織化学的検出によって、ミクログリア細胞内標的（ 例えば、Aβ）13-17。二つの研究は、 生体内でミクログリアの貪食作用を評価するために、より直接的なアプローチを使用してきました。ヒューズらは、頭蓋内経路18を介して配信ビーズのミクログリアの取り込みを測定するためのイメージング技術を使用しています。シエラらは、定量的に複雑なイメージング技術4を用いて、アポトーシス細胞のミクログリア食作用を評価するための洗練された方法を開発しました。しかしながら、これらの方法は、組織標本、セクショニング、画像化、および分析のための複雑なプロトコルを含みます。我々は以前外感光体の食作用を評価するためにフローサイトメトリー分析を使用しています文化19における網膜色素上皮（RPE）細胞による小胞体セグメント。ここでは、急速にin vivoでのミクログリアの貪食作用の定量的尺度として網膜ミクログリアにより、蛍光標識された粒子の取り込みを評価するためのプロトコルについて説明します。 蛍光標識された粒子の（1）硝子体内の配信、（2）収穫と網膜組織の準備、および（3）流量：私たちはここで説明するプロトコルは、信頼性が高く、定量的な3つの重要なステップですぐ下に6時間で網膜ミクログリア食作用の測定を可能にしますサイトメトリー分析。我々が開発した方法は網膜におけるミクログリア食作用を評価するための堅牢な方法であり、正常に種々の化合物または遺伝子操作は、生理学的設定でこのキーミクログリアの機能を変化させる方法をテストするために使用することができます。 CNSの専門分野として、網膜ミクログリア機能20を研究するための簡単にアクセスできるモデル系です。この方法は、トンで開発されたが彼の目、我々はそれがミクログリアの貪食機能を調査するすべての神経科学者のために役立つことができると信じています。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>Discontinued</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton&#160;syringe, 600 series</td>
			<td>Sigma</td>
			<td>26702</td>
			<td></td>
		</tr>
		<tr>
			<td>33 gauge, Small Hub RN NDL, 0.5 in, point style 4 - 12o</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymosan A (S. cerevisiae) BioParticles, Alexa Fluor 488 conjugate</td>
			<td>ThermoFisher Scientific</td>
			<td>Z-23373</td>
			<td>Prepare immediately before injection</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5/45 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
			<td>Need two</td>
		</tr>
		<tr>
			<td>Dumont #5SF Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors - 3mm Cutting Edge</td>
			<td>Fine Science Tools</td>
			<td>15000-10</td>
			<td>Curved</td>
		</tr>
		<tr>
			<td>Neural Tissue Dissociation Kit &#8211; Postnatal Neurons</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Polystyrene Round-bottom Tube</td>
			<td>Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well U-bottom plate</td>
			<td>Falcon</td>
			<td>353077</td>
			<td></td>
		</tr>
		<tr>
			<td>Stain Buffer (BSA)</td>
			<td>BD Biosciences</td>
			<td>554657</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b-BV650 Antibody</td>
			<td>BioLegend</td>
			<td>101259</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly6C-APC-Cy7</td>
			<td>BioLegend</td>
			<td>128025</td>
			<td></td>
		</tr>
		<tr>
			<td>Ly6G-PE-Cy7</td>
			<td>BioLegend</td>
			<td>127617</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>BD Biosciences</td>
			<td>556463</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified anti-mouse CD16/32 Antibody</td>
			<td>BioLegend</td>
			<td>101301</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

ここでは、この方法は、以下の貪食能の化合物および/または遺伝子操作の効果を試験するために適合させることができる（ 図2）を迅速かつ確実に、フローサイトメトリー分析を用いて、生理学的設定において食網膜ミクログリアの数を定量化する方法を記載しますミクログリア（ 図3A、3B）。 （ - 20日生後10）又は成体マウス（ <strong...

Watch this Scientific Journal Video about Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay at JoVE.com

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

組織の恒常性と不十分な食細胞機能の維持が病態に関与しているためにミクログリアの貪食作用が重要です。しかしながら、 インビボでのミクログリアの機能を評価することは技術的に困難です。私たちは、正確に監視し、生理的な環境の中でミクログリアの貪食の可能性を定量化するためのシンプルでありながら堅牢な技術を開発しました。

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, ...

The overall goal of this procedure is to assess the phagocytic function of retinal microglia in vivo. This method can help answer key questions in the ophthalmology field, such as whether certain compounds alter microglia phagocytic function in a physiological setting. The main advantage of this technique is that it uses flow cytometry, allowing a fast and precise quantitative analysis of microglial phagocytosis.Helping me to demonstrate the procedure will be Susumu Sakimoto, a postdoc from my laboratory. Begin by loading a 33-gauge needle and syringe with 0.5 microliters of fluorescently labeled particle solution. Then place an anesthetized mouse sideways on soft material under a surgical microscope and confirm the appropriate level of sedation by a lack of response to toe pinch.Next, use forceps to carefully apply pressure around one eyelid, so that the eye ball pops slightly out of the socket. Then grasp the head with two fingers just above the ear and by the animal's jaw, and gently stretch the skin parallel to the eyelids to keep the eye slightly bulged out of the socket. Intravitreal injections are challenging, and if not performed correctly will lead to biased and variable results.To reduce trauma to the eye, hold the animal in a stable position with minimal head movement. To puncture the eyeball, gently grasp the mouse with one hand. Then locate the corneal limbus where the cornea and sclera connect, which is visible as a gray circle in pigmented mice.Holding the syringe in the other hand, insert the needle into the limbus. Then retract the needle slightly to expel a small volume of vitreous fluid, and slowly depress the plunger to inject the particles. When all of the beads have been delivered, slowly withdraw the syringe to avoid reflux of the injected material and apply moisturizing drops to keep the eye hydrated once the eye has retracted back into place.Then place the animal on a heating pad in its own cage with monitoring until it is fully recovered. Three hours after intravitreal injection, use 45 degree angle forceps to gently press against the eyelid to proptose the eyeball. Position the forceps behind the eyeball and pull.Then transfer the eyeball to the dry area of a petri dish, containing a small volume of PBS with calcium and magnesium under a dissecting microscope. And use one tip of a superfine forceps to perforate the eye in the corneal limbus. Next, holding the eyeball with fine 45-degree angled forceps, use spring scissors to cut around the corneal limbus until roughly half of the circumference is cut.Transfer the eyeball into PBS and use a second pair of fine 45-degree angled forceps to tear the cornea and sclera apart. The lens and retina will come out intact. Ensure the lens and retina are separated and transfer the retina to a 5.4 milliliter polystyrene test tube, containing two milliliters of PBS with calcium and magnesium.To obtain a single cell suspension of the retinal cells, use a neuronal tissue dissociation kit according to the manufacturer's instructions and resuspend the cells in 200 microliters of staining buffer. Then transfer the sample to one well of a 96-well U-bottom plate. After centrifugation, invert the plate to discard the supernatant and block the FC receptors with 25 microliters of stain buffer containing CD16, CD32 antibody per well for five minutes at room temperature.Next label the cells with the antibodies of interest for 15 minutes at room temperature in the dark. Then pellet the cells and follow this by a wash in 200 microliters of fresh staining buffer per well. Now resuspend the pellets in 200 microliters of stain buffer and viability dye and transfer the samples to 1.2 milliliter microtiter tubes.Then wash the wells with an additional 100 microliters of staining buffer and viability dye and pool the washes with the corresponding samples for analysis by flow cytometry. This method can be used in 10 to 20-day old postnatal or adult mice and can be adapted to test the effect of compounds and/or the genetic manipulation of microglial phagocytosis. For example, in this experiment after intraperitoneal challenge with varying doses of LPS, a 1.42 milligram per kilogram dose of LPS was determined to induce a statistically significant increase in the percentage of phagocytic microglia, compared to vehicle challenged controls.Once mastered, this technique can be completed in six hours if it is performed properly. Following this procedure, other methods like cell sorting followed by qPCR or proteomic analysis, can be used to answer further questions about the differences between phagocytic and non phagocytic microglia in the retina. In developing this technique, it's our expectation that we can now make it possible for vision and neuro scientists to further explore microglial phagocytic function in situ and in a physiologically relevant context.

Research

JoVE Journal

Immunology and Infection

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

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

顆粒球機能の変化を評価するために、イメージベースのフローサイトメトリー技術<em&gt;インビトロ</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

免疫・感染学

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

NK細胞の機能を監視するためのサイトメトリーに基づくアッセイを流します

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

ヒト NK 細胞活性の評価の流れのフローサイトメトリーを用いた細胞毒性の試金

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

生体内で予防接種モデルを用いた抗原特異的 T 細胞細胞傷害性関数の検出のための試金

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

単純なフローサイトメトリーによるジカ回復期血清を用いたデング ウイルスのIn Vitro抗体依存強化測定法

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

ミトコンドリア、リソソーム T 細胞のマルチカラー フロー フローサイトメトリーを用いた定量化

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Assessing the Cellular Immune Response of the Fruit Fly, Drosophila melanogaster, Using an In Vivo Phagocytosis Assay

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ミバエショウジョウバエVivo で貪食能アッセイを使用しての細胞性免疫能を評価します。

In all animals, innate immunity provides an immediate and robust defense against a broad spectrum of pathogens. Humoral and cellular immune responses are ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

フローサイトメトリーベースアッセイによる熱帯 熱マラリア原 虫に対する自然獲得食細胞症誘導抗体の測定

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

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インフルエンザ菌に対する呼吸器免疫応答の評価

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells

フローサイトメトリーによる心筋B細胞の定量および分析(英語)

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. ...