Name: light-sheet microscopy for three-dimensional visualization of human immune cells
Uploaded: 2018-06-13T13:30:00
Description: Watch this Scientific Journal Video about Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells at JoVE.com

Vi takker Institutt for klinisk Hemostaseology og transfusjon medisin for å gi donor blod. Carmen Hässig og Cora Hoxha for utmerket teknisk hjelp. Vi takker Jens Rettig (Saarland University) for endret pMAX vektor, Roland Wedlich-Söldner (Universitetet i Muenster) for den opprinnelige LifeAct-Ruby konstruksjonen og Christian Junker (Saarland University) for å generere den LifeAct-mEGFP konstruksjonen. Dette prosjektet ble finansiert av Sonderforschungsbereich 1027 (prosjekt A2 til B.Q.) og 894 (prosjekt A1 til M.H.). Lys arks mikroskopet ble finansiert av DFG (GZ: INST 256/4 19-1 FUGG).

Forskning gjennomført denne undersøkelsen med menneskelige materialet (leukocytter reduksjon system chambers fra menneskeblod givere) er autorisert av den lokale etikk (erklæring fra 16.4.2015 (84/15; Prof Dr. Rettig-Stürmer)) og følger retningslinjene som er tilsvarende.
1. forberedelse men kollagen løsning (500 µL)
<ol><li>Overføre 400 µL av kjølt kollagen lagerløsning (10.4 mg/mL) til en bakteriefri 1.5 mL tube under celle kultur panseret. Sakte legge til 50 µL av kjølt 10 x ...

<ol>
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De fleste i vitro analyser utføres på 2D underlag, for eksempel i cellekultur plater, Petri-retter eller på coverslips, mens i vivo celler, spesielt immunceller, oppleve mest en 3D microenvironment. Nye bevis viser at migrasjon av immunceller varierer mellom 2D og 3D scenarier17. Videre er uttrykket profiler av kreftceller også annerledes i 2D - og 3D-kulturperler vev18,19,20. Derfor...

De fleste kunnskap om overføring cellene kommer fra 2D eksperimenter1,2,3, som normalt gjennomføres i et glass eller plast overflaten av kultur tenkelig parabolen. Imidlertid krever fysiologiske scenario i de fleste tilfeller en 3D microenvironment, der den ekstracellulære matrisen (EFM) spiller en avgjørende rolle. ECM ikke bare gir den 3D struktur avgjørende for å opprettholde riktig celle morfologi, men tilbyr også overlevelse signaler eller retningsbestemt signaler for en optimal funksjon av mange celler4,5 . Et 3D-miljø er derfor nødvendig å forbedre cellulære funksjoner og atferd i et miljø som er reflektere bedre fysiologisk sammenheng.
I menneskekroppen utøve de fleste celler spesielt immunceller, sine funksjoner under et 3D scenario. For eksempel aktivert T celler patruljere vev etter målcellene, naiv T celler vandrer gjennom lymfeknuter i Søk etter sine beslektet antigen-presentasjon celler der overføring modus og maskiner er tilpasset de tilsvarende ekstracellulære miljø3,6,7. 3D kollagen gel har vært mye brukt som en veletablert og godt karakterisert 3D celle kultur-systemet8,9,10. Våre tidligere arbeid viser at primære menneskelige lymfocytter er svært mobile og overføre med en gjennomsnittlig hastighet på rundt 4,8 µm/min i en 0.25% kollagen-baserte matrix11. Omorganisering av cytoskjelett spiller en nøkkelrolle i celle migrasjon12. Samler bevis viser at lymfocytter gjelder ikke bare et enkelt modus av migrasjon ennå kan bytte mellom visse migrasjon virkemåter avhengig av beliggenhet, microenvironment, cytokiner, chemotactic graderinger og ekstracellulære signaler som tune det vandrende atferd i ulike måter 3.
Pålitelig analysere immun celle funksjoner og atferd, for eksempel overføring, protrusion formasjon eller vesicula transport, er det stor fordel å kunne hente bilder i relativt store 3D volumer på en rask og pålitelig måte. For 3D-bildebehandling tilbyr nylig utviklet lys arks mikroskopi teknologien (også referert til som enkelt flyet belysning mikroskopi) en tilfredsstillende løsning13,14. Under bildebehandling oppkjøpet, genereres et tynt statisk lys ark for å belyse prøven. På denne måten, på fokus flyet, kan et stort område være opplyst samtidig uten at det påvirker av flyet cellene. Denne funksjonen kan en høy oppkjøpet hastighet med en drastisk redusert bleking og photocytotoxicity. I dette papir beskriver vi hvordan å visualisere primære menneskelige immunceller bruker lys arks mikroskopi og analysere overføringen i 3D scenario.

<table>
	<tbody>
		<tr>
			<td>Fibricol, bovine collagen solution</td>
			<td>Advanced Biomatrix</td>
			<td>&#160;#5133-20ML</td>
			<td>Collagen matrix</td>
		</tr>
		<tr>
			<td>0.5 M NaOH Solution</td>
			<td>Merck</td>
			<td>1091381000</td>
			<td>for neutralizing Fibricol solution</td>
		</tr>
		<tr>
			<td>Ultra-Low melting agarose</td>
			<td>Affymetrix</td>
			<td>32821-10GM</td>
			<td>Sample preparation in low c[Col]</td>
		</tr>
		<tr>
			<td>Dynabeads Untouched Human CD8 T Cells Kit</td>
			<td>Thermo Fisher</td>
			<td>11348D</td>
			<td>Isolation of primary human CD8+ T cells from PBMC</td>
		</tr>
		<tr>
			<td>Dynabeads Human T-Activator CD3/CD28 for T Cell Expansion and Activation</td>
			<td>Thermo Fisher</td>
			<td>11132D</td>
			<td>Activation of CTL populations</td>
		</tr>
		<tr>
			<td>Human recombinant interleukin-2</td>
			<td>Thermo Fisher</td>
			<td>PHC0023</td>
			<td>Stimulation of cultured CTL</td>
		</tr>
		<tr>
			<td>P3 Primary solution kit</td>
			<td>Lonza</td>
			<td>V4XP-30XX</td>
			<td>Transfection</td>
		</tr>
		<tr>
			<td>&#945;-PFN1 antibody, rabbit, IgG</td>
			<td>Abcam</td>
			<td>ab124904</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>Alexa Fluor 633 Phalloidin</td>
			<td>Thermo Fisher</td>
			<td>A22284</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>CellMask Orange Plasma membrane Stain</td>
			<td>Thermo Fisher</td>
			<td>C10045</td>
			<td>Fluorescent cell label</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P1379-250mL</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Eurobio</td>
			<td>018774</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>DPBS Dulbecco&#39;s phopsphate buffered saline</td>
			<td>Thermo Fisher</td>
			<td>14190250</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A9418-100G</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>Goat &#945; Rabbit 568, IgG, rabbit</td>
			<td>Thermo Fisher</td>
			<td>A-11011</td>
			<td>IF</td>
		</tr>
		<tr>
			<td>Lightsheet Z.1 (Light-sheet microscopy)</td>
			<td>Zeiss</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture hood</td>
			<td>Thermo Fisher</td>
			<td>HeraSafe KS</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator HERACell 150i&#160;</td>
			<td>Thermo Fisher</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5418 and 5452</td>
			<td>Eppendorf</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippettes</td>
			<td>Eppendorf</td>
			<td>3123000039, 3123000020, 3123000063</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippette tips</td>
			<td>VWR</td>
			<td>89079-444, 89079-436, 89079-452&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Sarstedt</td>
			<td>&#160;62.554.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillaries 50 &#181;L</td>
			<td>VWR (Brand)</td>
			<td>613-3373</td>
			<td>Zeiss LSFM sample preparation</td>
		</tr>
		<tr>
			<td>Plunger for capillaries</td>
			<td>VWR (Brand)</td>
			<td>BRND701934</td>
			<td>&#34;Stamps with Teflon tip&#34; LSFM sample preparation</td>
		</tr>
		<tr>
			<td>MColorPhast pH stips</td>
			<td>Merck</td>
			<td>1095430001</td>
			<td>to test pH of neutralized Fibricol</td>
		</tr>
		<tr>
			<td>BD Plastipak 1mL syringes</td>
			<td>BD</td>
			<td>Z230723&#160;ALDRICH</td>
			<td>Alternative sample preparation</td>
		</tr>
		<tr>
			<td>Modeling clay (Hasbro Play-Doh A5417EU7)</td>
			<td>Play-Doh</td>
			<td>N.A.</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris file converter</td>
			<td>Bitplane</td>
			<td>available at http://www.bitplane.com&#160;</td>
			<td>Convert imaging files to Imaris file format</td>
		</tr>
		<tr>
			<td>Imaris 8.1.2 (MeasurementPro, Track, Vantage)</td>
			<td>Bitplane</td>
			<td>available at http://www.bitplane.com&#160;</td>
			<td>Analysis of 3D and 4D imaging data</td>
		</tr>
	</tbody>
</table>

light-sheet microscopy for three-dimensional visualization of human immune cells

I vivo, aktivisering, spredning og funksjon av immunceller alle oppstå i et tredimensjonalt (3D)-miljø, for eksempel i lymfeknuter eller vev. Oppdatert avhengige de fleste i vitro systemer av todimensjonal (2D) overflater, for eksempel cellekultur plater eller coverslips. Optimalt etterligne fysiologiske forhold i vitrobruker vi en enkel 3D kollagen matrise. Kollagen er en av hovedkomponentene i ekstracellulær matrix (ECM) og har vært mye brukt å utgjøre 3D matriser. For 3D-bildebehandling, er nylig utviklet lys arks mikroskopi teknologien (også referert til som enkelt flyet belysning mikroskopi) kjennetegnet med høy oppkjøpet hastighet, store gjennomtrenging dyp, lav bleking og photocytotoxicity. Videre er lys-arks mikroskopi særlig fordelaktig for langsiktige mål. Her beskriver vi en optimalisert protokoll hvor sette og håndtere menneskelige immunceller, f.eks primære menneskelige cytotoksiske T-lymfocytter (CTL) og naturlig killer (NK) celler i 3D kollagen matrisen for bruk med lys arks mikroskopi for levende celle bildebehandling og fast prøver. Fremgangsmåten for bildeopptak og analyse av celle migrasjon presenteres. Et særlig fokus er gitt til markere avgjørende skritt og for eksempel forberedelse og dataanalyse. Denne kan benyttes for andre typer suspensjon celler i en 3D kollagen matrise og er ikke begrenset til immunceller.

Protrusion formasjon under T celle migrasjon er en svært dynamisk prosess, som er begrepsordbok avhengige. For å visualisere protrusion dannelsen av primære menneskelige CTL, transfected vi transiently en mEGFP smeltet protein Hvis etiketten begrepsordbok cytoskeleton i CTLEN som beskrevet før11. En dag etter transfection, var cellene innebygd i kollagen matrisen. Bildestakker anskaffet hver 40 s trinn-størrelse på 1 µm ved 37 ° C med lys arks mikroskopi. S...

Watch this Scientific Journal Video about Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells at JoVE.com

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

Lys arks mikroskopi for tredimensjonale visualisering av menneskelig immunceller

Her presenterer vi en protokoll for å visualisere immunceller i en tredimensjonal (3D) kollagen matrise med lys arks mikroskopi. Denne protokollen også utdyper hvordan spore celle migrasjon i 3D. Denne protokollen kan benyttes for andre typer suspensjon celler i 3D matrix.

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

Research

JoVE Journal

Immunology and Infection

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

Seven Steps to Stellate Cells

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Intravital Imaging av Mouse Thymus med 2-Photon Mikroskopi

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion (STED) Nanoscopy

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated Emission Depletion STED Nanoscopy

Visualisering av den immunologiske Synapse av Dual Color Time-gated Stimulert Emission Depletion (STED) Nanoscopy

Natural killer cells form tightly regulated, finely tuned immunological synapses (IS) in order to lyse virally infected or tumorigenic cells. Dynamic ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Long Term intra multiphoton Mikros Imaging av immunceller i friske og syke leveren Bruke CXCR6.Gfp Reporter Mus

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

En semi-automatisert tilnærming til Klar Antistoff Cocktails for Immunophenotypic analyse av perifert blod

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Kvantifisering av Efferocytosis av encellede fluorescens mikroskopi

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Three-dimensional Imaging of Bacterial Cells for Accurate Cellular Representations and Precise Protein Localization

Tredimensjonal Imaging av bakterielle celler for nøyaktig Cellular representasjoner og presis protein lokalisering

The shape of a bacterium is important for its physiology. Many aspects of cell physiology such as cell motility, predation, and biofilm production can be ...