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.

Alle dyrene ble behandlet i henhold til de etiske retningslinjene fastsatt av Scripps Research Institute. 1. Utarbeidelse av materialer for injeksjon <ol><li> Sterilisere en 33 G nål og sprøyte: demontere og autoklav ved 115 ο C. Forbered nåler for injeksjon av stigende i steril fosfat saltvann (PBS). </li><li> Tine fluorescensmerkede partikler ved romtemperatur i 5 - 10 min. Fremstille partikkelinjeksjonsvæske ved rekonstituering til en 50 mg / ml oppløsning i sterilt PBS med Ca2 +...

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Det er tre kritiske trinn i denne fremgangsmåten: (1) intravitreal injeksjon av fluorescensmerkede partikler; (2) høsting og forberedelse av retinal vev; og (3) flowcytometrisk analyse. Vi anbefaler at forskere øve intravitreale injeksjoner før gjennomføring av den metoden vi presenterer her. Albino-mus (for eksempel Balb / c) og en farget oppløsning (for eksempel fluorescerende merkede partikler) kan benyttes for enkel visualisering av nål og injisert løsning. Intravitreale injeksjoner er utfo...

Det overordnede målet med denne metoden er å nøyaktig vurdere og kvantifisere in vivo microglial fagocytose. Microglia er vevet bosatt makrofager i sentralnervesystemet (CNS). De utfører en rekke funksjoner for å sikre vedlikehold av vev homeostase. Disse inkluderer immune overvåking, sekresjon av neurotrofiske faktorer og, avgjørende betydning, fagocytose en. Microglial fagocytose er nøkkelen i flere viktige hendelser i utviklingen av hjernen og netthinnen, som for eksempel fagocytose av irrelevante synapser (synaptiske beskjæring) og fjerning av apoptotiske nevroner 2-4. Videre har microglial fagocytose av skadede eller apoptotiske nevroner, cellerester og invaderende mikrober vist seg å være avgjørende for å opprettholde CNS homeostase gjennom voksenlivet fem. Endelig har microglial fagocytose blitt implisert i patogenesen av en rekke neurodegenerative sykdommer, inkludert Alzheimers sykdom og aldersrelatertmakuladegenerasjon, hvor det har vært antydet at defekte eller manglende fagocytisk kapasitet kan bidra til oppbygging av amyloid-beta (Ap) plaques og drusen, henholdsvis 6,7. Microglial funksjon er strengt regulert av deres mikromiljø, særlig av løselige faktorer, slik som tumor-vekstfaktor β eller celle-celle interaksjoner. Nerveceller konstitutivt uttrykke flere celleoverflate ligander, for eksempel CD200 og CX3CL1, mens microglia utelukkende uttrykker de respektive reseptorer CD200R og CX3CR1. Disse reseptorene inneholder immunoreceptor tyrosin-basert hemming motiver (ITIMs) i sin intracellulær del. Disse inhibitor reseptorer er kritisk for å hindre overstimulering av mikroglia, noe som kan bidra til nevroinflammasjon. Således, under normale fysiologiske betingelser, celle-celle interaksjoner mellom nevroner og mikroglia holde mikroglia i en hviletilstand. Under vevsskade, men nevroner kan nedregulerer expression av disse ligandene, fjerning av deres hemmende virkning på mikroglia aktivering. Microglial funksjon (inkludert fagocytose) blir dermed tett knyttet til deres mikromiljøet 8. Likevel, til dags dato, er det ingen standardiserte analyser for å studere microglia fagocytose i en fysiologisk sammenheng eller på en måte som fullt gjenskaper sin CNS mikromiljøet. Flere analyser er blitt utviklet for å måle fagocytisk aktivitet av mikroglia in vitro, hvor primær mikroglia eller microglia cellelinjer blir dyrket med målceller (f.eks apoptotiske neuroner) eller fluorescerende merkede kuler. Target opptak blir deretter vurdert ved hjelp av fluorescerende bildebehandling mikroskopi eller flowcytometri 9-12. Disse analysene tillate testing av hvordan farmakologisk eller genetisk manipulasjon kan påvirke microglial fagocytose og mens informativ, ikke klarer å full replikere komplekse in vivo miljø. Indirekte metoder for å undersøke microglial fagocytosein vivo er rapportert: disse er oppnådd ved farging av molekyler antas å være involvert i fagocytose (f.eks CD68), vurdere fysisk nærhet av microglia og mål for fagocytose (f.eks kompromittert nevroner eller synaptic elementer), eller ved immunhistokjemisk påvisning av fagocytisk mål innenfor mikrogliaceller celler (f.eks Ap) 13-17. To studier har brukt mer direkte tilnærminger for å vurdere microglia fagocytose in vivo. Hughes og kolleger har brukt bildeteknikker for å måle microglial opptak av perler som leveres via intrakranielle rute 18. Sierra et al. Utviklet en raffinert metode for å vurdere kvantitativt microglia fagocytose av apoptotiske celler ved hjelp av komplekse bildeteknikker 4. Men disse metodene involverer kompliserte protokoller for vev forberedelse, seksjonering, bildebehandling og analyse. Vi har tidligere brukt flowcytometrisk analyse for å vurdere fagocytose av fotoreseptoren uteere segmenter av netthinnens pigmentert epitel (RPE) celler i kultur 19. Her beskriver vi en protokoll for raskt å vurdere opptak av fluorescensmerkede partikler med retinal mikroglia som et kvantitativt mål på in vivo mikroglia fagocytose. Protokollen vi beskriver her gir mulighet for pålitelig og kvantitativ måling av retinal microglial fagocytose i underkant av seks timer i tre kritiske trinn: (1) intravitreal levering av fluorescensmerket partikler, (2) høsting og utarbeidelse av retinal vev, og (3) flyt cytometri analyse. Metoden vi har utviklet er en robust metode for å vurdere microglial fagocytose i netthinnen, og det kan med hell brukes til å teste hvordan forskjellige forbindelser eller genetisk manipulasjon endre denne nøkkelen microglial funksjonen i fysiologiske innstillinger. Som et spesialisert område av CNS, er netthinnen en lett tilgjengelig modellsystem for å studere mikroglia funksjon 20. Selv om denne metoden ble utviklet i than øye, tror vi det kan være nyttig for alle nevrologer undersøker microglia fagocyttfunksjon.

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			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>Discontinued</td>
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			<td>Hamilton&#160;syringe, 600 series</td>
			<td>Sigma</td>
			<td>26702</td>
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			<td>33 gauge, Small Hub RN NDL, 0.5 in, point style 4 - 12o</td>
			<td>Hamilton</td>
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			<td>Zymosan A (S. cerevisiae) BioParticles, Alexa Fluor 488 conjugate</td>
			<td>ThermoFisher Scientific</td>
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			<td>Vannas Spring Scissors - 3mm Cutting Edge</td>
			<td>Fine Science Tools</td>
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			<td>Curved</td>
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			<td>Neural Tissue Dissociation Kit &#8211; Postnatal Neurons</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-802</td>
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			<td>5 mL Polystyrene Round-bottom Tube</td>
			<td>Falcon</td>
			<td>352054</td>
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			<td>96 well U-bottom plate</td>
			<td>Falcon</td>
			<td>353077</td>
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			<td>Stain Buffer (BSA)</td>
			<td>BD Biosciences</td>
			<td>554657</td>
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			<td>BioLegend</td>
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			<td>BioLegend</td>
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			<td>Propidium Iodide</td>
			<td>BD Biosciences</td>
			<td>556463</td>
			<td></td>
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			<td>Purified anti-mouse CD16/32 Antibody</td>
			<td>BioLegend</td>
			<td>101301</td>
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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.

Her beskriver vi en fremgangsmåte til hurtig og pålitelig måte å kvantifisere antallet fagocyterende retinal mikroglia i et fysiologisk miljø ved bruk av strømningscytometrisk analyse (Figur 2). Denne fremgangsmåten kan tilpasses for å teste effekten av forbindelser og / eller genetisk manipulasjon på den fagocytisk kapasitet mikroglia (figurene 3A, 3B). Den kan også brukes i unge (10 - 20 dager etter fødselen) eller voksen mus <stron...

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

Microglial fagocytose er avgjørende for vedlikehold av vev homeostase og utilstrekkelig fagocyttfunksjon har vært innblandet i patologi. Imidlertid vurdering av mikroglia funksjon in vivo er teknisk utfordrende. Vi har utviklet en enkel men robust teknikk for nøyaktig å overvåke og kvantifisering av fagocytisk potensialet av mikroglia i et fysiologisk miljø.

Vurdere Netthinne microglial fagocyttfunksjon<em&gt; I Vivo</em&gt; Ved hjelp av en flowcytometrisystemer basert analyse

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

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JoVE Journal

Immunology and Infection

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

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Vurdere Medfødt Sensing av HIV-1 infiserte CD4<sup&gt; +</sup&gt; T-celler ved Plasmacytoid dendrittiske celler ved hjelp av en<em&gt; Ex vivo</em&gt; Co-kultursystem.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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

Bildebasert flowcytometrisystemer Technique å evaluere Endringer i Granulocytt Funksjon<em&gt; In Vitro</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 ...

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

Flowcytometri basert analyse for overvåking av NK cellefunksjoner

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

En flyt cytometri-baserte cytotoksisitet analysen for vurdering av menneskelig NK celle aktivitet

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

I Vivo Analysen for påvisning av Antigen-spesifikke T-celle Cytolytic funksjon med en vaksinasjon modell

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

En enkel flowcytometri basert analyse for å fastslå In Vitro antistoff avhengige forbedring av Dengue Virus benytter Zika Virus rekonvalesent Serum

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

Flerfarget flyt cytometri-baserte kvantifisering av mitokondrier og lysosomer i T-celler

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

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

Vurdere mobilnettet immunforsvaret frukt fly, Drosophila melanogaster, bruke i Vivo fagocytose analysen

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

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain

Immunofluorescence farging bruke IBA1 og TMEM119 for Mikrogliaaggregater Density, morfologi og perifere myelogen Cell infiltrasjon analyse i Mouse Brain

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia ...

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

Måling naturlig ervervet phagocytosis-induserende antistoffer mot Plasmodium falciparum parasitter ved en Flow Cytometry-basert analyse

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