Name: sample preparation and imaging of exosomes by transmission electron microscopy
Uploaded: 2018-01-04T13:00:00
Description: Watch this Scientific Journal Video about Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy at JoVE.com

Denne forskningen ble støttet av Bio &#38; medisinsk teknologi Development Program av National Research Foundation (NRF) &#38; finansiert av koreanske myndigheter (MSIP &#38; MOHW) (nr. 2016M3A9B6904244). Vi takker også medlemmer av Medisintran Bioconvergence Research Center for rensing av exosome.

1. kvartal forberedelse, skjæring, flekker og Imaging av Exosome
<ol>
	<li>Pellets exosomes fra kultur nedbryting av HCT116 celler med sentrifugering 100.000 x g for 1,5 t23. Fjerne kulturen supernatant og nøye løse renset exosome pellets med 1 mL 2,5% glutaraldehyde i 0.1 M natrium cacodylate løsning (pH 7.0) 1t på 4 ° C. For 0.1 M natrium cacodylate, løses 4,28 g cacodylic syre i 160 mL destillert vann (DW). Justere pH 7,4 med 0,1 M HCl gjør deretter opp til 200 mL med destillert vann....

<ol>
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Denne artikkelen presenterer en protokoll for å observere det detaljert exosome struktur og merking av sine spesifikke proteiner. Negativ flekker har vært ansett som den beste metoden for exosome imaging17. Denne konvensjonelle teknikken vist exosomes' cup-formet struktur. Men er denne kopp form en form for gjenstanden som kan skyldes tørkeprosessen. Cryo-TEM resultatene har vist at exosomes har en perfekt sfærisk struktur i vandig løsning25,<sup class="xref...

Ekstracellulære blemmer (EVs) er lipid bilayer blemmer utskilles av celler, og deres størrelse varierer mellom 30 og 300 nm. EVs ble først rapportert i 1978 med bevis fra blemmer av pasienter med Hodgkins sykdom1. Disse vesikler ble rapportert å være 40 til 120 nanometer i størrelse. I 1980 ble det rapportert at EVs er involvert i koagulasjonssystemet og blodpropp, dannelse av blodpropp i en blodåre i kreft pasienter2. Etter 30 år, er EVs rapportert å være en viktig faktor å fremme svulst invasjon, immun rømme og angiogenese3. Dessuten, har funksjonene til EVs studert som regulatorer i mobilnettet interaksjoner i områder av betennelse, immune disorder, nevrologisk sykdom og kreft4. Siden EVs inneholder bestemte biomolecules som protein, mRNA og microRNA5, har deres potensielle programmet i diagnostikk og therapeutics vært analysert6,7. EVs er en kategori består av undergrupper inkludert exosomes, prostasomes, oncosomes, dexosomes, microparticles, promininosomes, argosomes og exosome som blemmer, avhengig av mobilnettet opprinnelse og biologisk funksjon3. I tillegg kan basert på deres biogenesis, disse EVs deles inn i microvesicles (skur microvesicles, 100-1000 nm) og exosomes (30-300 nm)8,9. Blant disse, har exosome blitt rapportert som en celle kommunikator for immunforsvaret10, kreft11,12og smittsomme sykdommer13.
Interesse i exosomes som en biomarkør for tidlig diagnostisering øker raskt, og rensing og karakterisering av exosomes må ledsages med molekylær Bildeteknikker. Ulik størrelse og morphologies av exosomes, avhengig av deres opprinnelse og funksjonen14, kan være preget av mikroskopi teknikker med høy oppløsning, for eksempel elektronmikroskop. De fleste exosomes var visualisert ved negative farget overføring elektronmikroskop (TEM)15,16,17, og disse resultatene ble bekreftet av immunolabeling av et bestemt protein i hele mount vesicle 18. flere forskningsgrupper har rapportert strukturen gjennom skanning elektronmikroskop, Atomic Force mikroskopi19,20og Cryo-TEM21,22. Men mens disse teknikkene er nyttige for å studere exosome strukturen, er de ikke nok for å observere plasseringen av spesifikke proteiner ligger inne på exosome. Derfor innført vi en protokoll for imaging exosomes med et bestemt protein merking. Vi brukt blokk forberedelse, supertynn snitting og immunostai-for konstatere protein's detaljert beliggenhet i exosome. Dette ble sammenlignet med negative flekker og hele mount immunostai-, som tradisjonelt brukes for karakteristikk av exosome.

<table border="0" cellpadding="0" cellspacing="0" width="583">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Glutaraldehyde</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">16200</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium cacodylate&#160;</td>
			<td>EMS</td>
			<td style="width:119px;">12300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Osmium tetroxide 1 g crystal</td>
			<td>EMS</td>
			<td style="width:119px;">19100</td>
			<td>Use only in fume hood</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">acetone</td>
			<td>JUNSEI</td>
			<td>1B2031</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Spurr medium</td>
			<td>TED PELLA</td>
			<td style="width:119px;">18108</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Ultra-microtome</td>
			<td style="width:81px;">Leica</td>
			<td style="width:119px;">UCT</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Uranyl acetate</td>
			<td>EMS</td>
			<td style="width:119px;">22400</td>
			<td>Hazardous chemical</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Lead citrate</td>
			<td>EMS</td>
			<td style="width:119px;">17900</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Transmission Electron Microscopy</td>
			<td style="width:81px;">Hitachi</td>
			<td style="width:119px;">H7600</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">nickel grid</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">G200-Ni</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Copper grid</td>
			<td>EMS</td>
			<td style="width:119px;">G200-Cu</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">glycine</td>
			<td>SIGMA</td>
			<td>022K5404</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Phosphate buffer saline</td>
			<td style="width:81px;">SIGMA</td>
			<td style="width:119px;">P4417</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Bovine serum albumin</td>
			<td style="width:81px;">Aurion</td>
			<td style="width:119px;">25557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">1st Antibody</td>
			<td style="width:81px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">purification in manually&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Purified anti-human CD274 (B7-H1, PD-L1) Antibody</td>
			<td style="width:81px;">BioLegend</td>
			<td style="width:119px;">329710</td>
			<td style="width:167px;">Whole mount Immumogold</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Purified Mouse IgG2b, &#954; Isotype Ctrl</td>
			<td style="width:81px;">BioLegend</td>
			<td style="width:119px;">401202</td>
			<td style="width:167px;">Whole mount Immumogold (Control)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">2nd Anti-mouse igG conjugated 9-11 nm gold particle</td>
			<td style="width:81px;">sigma</td>
			<td style="width:119px;">G7652</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">silver paint</td>
			<td style="width:81px;">CANS</td>
			<td style="width:119px;">CTP100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">aluminum stub</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">75622</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">FIB-SEM</td>
			<td style="width:81px;">Zeiss</td>
			<td style="width:119px;">AURIGA</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Transmission Electron Microscopy</td>
			<td style="width:81px;">JEOL</td>
			<td style="width:119px;">JEM2200FS</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Glow discharger</td>
			<td style="width:81px;">JEOL</td>
			<td style="width:119px;">JFC1100E</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Carbon grid</td>
			<td>EMS</td>
			<td style="width:119px;">121119</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Paraformaldehyde</td>
			<td>EMS</td>
			<td style="width:119px;">19210</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Formvar carbon coated Copper Grid (200 meh)</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">FCF200-CU</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Formvar carbon coated&#160; Nickel Grid (200 mesh)</td>
			<td style="width:81px;">EMS</td>
			<td style="width:119px;">FCF200-NI</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

sample preparation and imaging of exosomes by transmission electron microscopy

Exosomes er nano-størrelse ekstracellulære blemmer utskilles av kroppsvæsker, og er kjent som representerer egenskapene til celler det avsondre dem. Innholdet og morfologi av secreted blemmer gjenspeile cellen atferd eller fysiologiske status, for eksempel cellevekst, migrasjon, cleavage og død. Exosomes' rolle kan svært avhengig av størrelse og størrelsen på exosomes varierer fra 30 til 300 nm. Den mest brukte metoden for exosome imaging er negativ flekker, mens andre resultater er basert på Cryo-overføring elektronmikroskop og skanning elektronmikroskop Atomic Force mikroskopi. Den typiske exosome morfologi vurdert gjennom negative flekker er en kopp-form, men ytterligere detaljer er ikke ennå klar. En exosome godt karakterisert gjennom strukturelle studien er nødvendig spesielt i medisinske og farmasøytiske. Derfor må funksjon-avhengige morfologi verifiseres av elektronmikroskop teknikker som merking et bestemt protein i detaljert strukturen i exosome. For å observere detaljert struktur, ble supertynn valgte bilder og negative farget bilder av exosomes sammenlignet. I denne protokollen foreslår vi overføring elektronmikroskop for avbilding av exosomes inkludert negative flekker, hele mount immuno-flekk, blokk forberedelse, tynne delen og immun-gull merking.

Foreløpig er exosomes klassifisert i størrelse og form kategorier av overføring elektronmikroskop. Figur 2 viser negative farget exosome og immun-merket exosome i hele mount status. Figur 3 viser valgte exosome og immun-merket exosomes etter tynn snitting. Immuno-gull farging med antistoffer spesifikke proteiner brukes å positiv identifisere en exosome og klassifisere typen proteiner i exosome. Protokollen i dette dokumentet ...

Watch this Scientific Journal Video about Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy at JoVE.com

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Eksempel forberedelse og bildebehandling for Exosomes av overføring elektronmikroskop

Denne protokollen beskriver ulike teknikker nødvendig for overføring elektronmikroskop inkludert negative flekker, supertynn snitting for detaljert struktur og immun-gull merking for å bestemme plasseringen av spesifikke proteiner i exosomes.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Research

JoVE Journal

Biology

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Prøvepreparering for mikroskopi av enkeltvirionakroskopi og superoppløselig fluorescensavbildning

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Cryo-elektron mikroskopi prøven forberedelse ved hjelp av en Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Sporing av Hematopoietiske Stem og stamceller i Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Tandem Høytrykks Frysing og Quick Freeze Bytte av plantemateriale for transmisjonselektronmikroskopi

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Gjøre og ikke gjøre av Cryo-elektronmikroskopi: En Primer på Prøvepreparering og High Quality datainnsamling for Macromolecular 3D Rekonstruksjon

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

Utarbeidelse og Observasjon av Thick biologiske prøver av Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Miniatyriserte eksempel forberedelse til overføring elektronmikroskop

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

Biologiske utvalg forberedelse av høytrykks fryser, mikrobølgeovn-assistert kontrast ekstrautstyr og Minimal harpiks innebygging volum Imaging

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...