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 Institut for klinisk Hemostaseology og Transfusion medicin for at give donor blod; Carmen Hässig og Cora Hoxha for fremragende teknisk hjælp. Vi takker Jens Rettig (Saarland Universitet) for den modificerede pMAX vektor, Roland Wedlich-Söldner (University Muenster) for de oprindelige LifeAct-Ruby konstruktion og Christian Junker (Saarland Universitet) til at generere LifeAct-mEGFP-konstruktionen. Dette projekt blev finansieret af Sonderforschungsbereich 1027 (projekt A2 til B.Q.) og 894 (M.H. projekt A1). Lys-ark mikroskopet blev finansieret af DFG (GZ: INST 256/4 19-1 FUGG).

Forskning udført for denne undersøgelse med det menneskelige materiale (leukocyt reduktion system kamre fra menneskelige bloddonorer) er godkendt af den lokale etiske komité (erklæring fra 16.4.2015 (84/15; Prof. Dr. Rettig-Stürmer)) og følger retningslinjerne for tilsvarende.
1. forberedelse af neutraliseret kollagen løsning (500 µL)
<ol><li>Overføre 400 µL af kølet kollagen stamopløsning (10.4 mg/mL) til en sterile 1,5 mL tube under celle kultur hætte. Tilsættes langsomt 50 ?...

<ol>
	<li>Decaestecker, C., Debeir, O., Van Ham, P., Kiss, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+anti-migratory+drugs+be+screened+in+vitro?+A+review+of+2D+and+3D+assays+for+the+quantitative+analysis+of+cell+migration.">Can anti-migratory drugs be screened in vitro? A review of 2D and 3D assays for the quantitative analysis of cell migration.</a> Med Res Rev. 27 (2), 149-176 (2007).</li><li>Doyle, A. D., Petrie, R. J., Kutys, M. L., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimensions+in+cell+migration.">Dimensions in cell migration.</a> Curr Opin Cell Biol. 25 (5), 642-649 (2013).</li><li>Krummel, M. 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J., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=At+the+leading+edge+of+three-dimensional+cell+migration.">At the leading edge of three-dimensional cell migration.</a> J Cell Sci. 125 (Pt 24), 5917-5926 (2012).</li><li>Imamura, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+2D-+and+3D-culture+models+as+drug-testing+platforms+in+breast+cancer).">Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer).</a> Oncol Rep. 33 (4), 1837-1843 (2015).</li><li>Lee, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+microenvironment+alters+protein+expression+and+chemosensitivity+of+epithelial+ovarian+cancer+cells+in+vitro.">A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro.</a> Lab Invest. 93 (5), 528-542 (2013).</li><li>Luca, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+the+3D+microenvironment+on+phenotype,+gene+expression,+and+EGFR+inhibition+of+colorectal+cancer+cell+lines.">Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines.</a> PLoS One. 8 (3), e59689 (2013).</li><li>Jemielita, M., Taormina, M. J., Delaurier, A., Kimmel, C. B., Parthasarathy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+phototoxicity+during+the+development+of+a+zebrafish+craniofacial+bone+using+confocal+and+light+sheet+fluorescence+microscopy+techniques.">Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.</a> J Biophotonics. 6 (11-12), 920-928 (2013).</li><li>Graf, B. W., Boppart, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+analysis+of+three-dimensional+cell+culture+models.">Imaging and analysis of three-dimensional cell culture models.</a> Methods Mol Biol. 591, 211-227 (2010).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat Methods. 2 (12), 932-940 (2005).</li><li>Huisken, J., Swoger, J., Del Bene, F., Wittbrodt, J., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+deep+inside+live+embryos+by+selective+plane+illumination+microscopy.">Optical sectioning deep inside live embryos by selective plane illumination microscopy.</a> Science. 305 (5686), 1007-1009 (2004).</li><li>Verveer, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+three-dimensional+imaging+of+large+specimens+with+light+sheet-based+microscopy.">High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy.</a> Nat Methods. 4 (4), 311-313 (2007).</li><li>Truong, T. V., Supatto, W., Koos, D. S., Choi, J. M., Fraser, S. 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De fleste in vitro- assays er udført på en 2D overflade, for eksempel i cellekultur plader, petriskåle eller på coverslips, i vivo celler, især immunceller, har for det meste en 3D mikromiljø. Nye beviser viser, at migrationsmønstre af immunceller varierer mellem 2D- og 3D-scenarier17. Desuden er udtrykket profiler af tumorceller er også forskellige i 2D - og 3D-kulturperler væv18,19,...

De fleste viden om overflytning celler kommer fra 2D eksperimenter1,2,3, som foregår normalt i et glas eller plastik overfladen af en kultur/imaging parabol. En fysiologisk scenario kræver dog i de fleste tilfælde, en 3D mikromiljø, hvori den ekstracellulære matrix (ECM) spiller en afgørende rolle. ECM ikke blot giver 3D-struktur er væsentlige for at opretholde ordentlig celle morfologi, men tilbyder også overlevelse signaler eller retningsbestemt stikord til en optimal funktion af mange celler4,5 . Et 3D-miljø er derfor påkrævet til bedre identificere cellulære funktioner og adfærd i et miljø, som bedre afspejler den fysiologiske kontekst.
I den menneskelige krop udøve de fleste celler især immunceller, deres funktioner under en 3D scenario. For eksempel, aktiverede T celler patruljere væv søger target-cellerne, naive T celler vandrer gennem lymfeknuder i søgen efter deres beslægtet antigen-præsenterer celler hvor overførselstilstanden og maskiner er tilpasset til de tilsvarende ekstracellulære miljø3,6,7. 3D kollagen gel har været udbredt som en veletableret og godt karakteriseret 3D celle kultur system8,9,10. Vores tidligere arbejde viser, at primære humane lymfocytter er meget mobile og overflytte ved en gennemsnitlig hastighed på omkring 4,8 µm/min med en 0,25% kollagen-baserede matrix11. Omlægning af cytoskeleton spiller en central rolle i celle migration12. Akkumulere beviser viser, at lymfocytter gælder ikke kun en enkelt transportform migration endnu kan skifte mellem visse migration opførsel afhængigt af placering, mikromiljø, cytokiner, kemotaktisk forløb, og ekstracellulære signalerer, hvilken melodi det vandrende adfærd i forskellige måder 3.
Til at pålideligt analysere immun cellefunktioner og adfærd, for eksempel, migration, fremspring dannelse eller vesikulær transport, er det af stor fordel at kunne erhverve billeder i forholdsvis stor 3D diskenheder på en hurtig og pålidelig måde. For 3D imaging giver nyligt udviklede lys-ark mikroskopi teknologi (også kaldet enkelt fly belysning mikroskopi) en tilfredsstillende løsning13,14. Under imaging erhvervelse, genereres en statisk lys tyndplade skal belyse prøven. På denne måde, i fokus flyet, belyst et stort område samtidigt uden at påvirke de off-plane celler. Denne funktion giver mulighed for en høj erhvervelse hastighed med en drastisk reduceret blegning og fototoksicitet. I dette papir beskriver vi hvordan man visualisere primære human immun celler ved hjælp af lys-ark mikroskopi og hvordan man kan analysere migration i en 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

In vivo, aktivering, spredning og funktionen af immun celler alle forekomme i en tre-dimensionelle (3D) miljø, for eksempel i lymfeknuder eller væv. Op til dato stole de fleste in vitro- systemer på todimensionelle (2D) overflader, såsom cellekultur plader eller coverslips. Til optimalt efterligne fysiologiske tilstande i vitrobenytter vi en simpel 3D kollagen matrix. Kollagen er et af de vigtigste bestanddele af ekstracellulære matrix (ECM) og har været meget anvendt til at udgøre 3D matricer. For 3D imaging, er den nyligt udviklede lys-ark mikroskopi teknologi (også kaldet enkelt fly belysning mikroskopi) featured med høje anskaffelses hastighed, stor indtrængningsdybde, lav blegning og fototoksicitet. Desuden er lys-ark mikroskopi særligt fordelagtigt for langsigtet måling. Her vi beskriver en optimeret protokol, hvordan til at oprette og håndtere humane immun celler, fx primære menneskelige cytotoksiske T-lymfocytter (CTL) og natural killer (NK) celler i 3D kollagen matrix til brug med lys-ark mikroskopi for levende celle imaging og faste prøver. Proceduren for image erhvervelse og analyse af celle migration er præsenteret. Særligt fokus er givet at fremhæve kritiske trin og faktorer til forberedelse af prøver og dataanalyse. Denne protokol kan være ansat af andre former for affjedring celler i en 3D kollagen matrix og er ikke begrænset til immunceller.

Fremspring dannelse under T-celle migration er en yderst dynamisk proces, som er actin afhængige. For at visualisere fremspring dannelsen af primær humane Tillidsliste, transfekteret vi forbigående en mEGFP af smeltet protein til at mærke actin cytoskelettet i CTL som beskrevet før11. En dag efter Transfektion, blev cellerne integreret i kollagen matrix. Billedstakke blev erhvervet hver 40 s med en trin-størrelse på 1 µm ved 37 ° C ved hjælp af lys-ark mi...

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-ark mikroskopi for tre-dimensionelle visualisering af Human immun celler

Vi præsenterer her, en protokol for at visualisere immunceller indlejret i en tre-dimensionelle (3D) kollagen matrix ved hjælp af lys-ark mikroskopi. Denne protokol også uddyber hvordan man spore celle migration i 3D. Denne protokol kan være ansat af andre former for affjedring celler i den 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 af Mouse Thymus med 2-Photon Microscopy

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

Visualiseringen af ​​det immunologiske Synapse med Dual Color Time-gated stimuleret emission Nedbrydning (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 Intravital multiphoton Microscopy Imaging af immunceller i rask som syg Lever Brug 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-automatiske tilgang til Forberedelse Antibody Cocktails for immunfænotypisk Analyse af Human Perifer 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

Kvantificering af Efferocytosis af 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

Tredimensionel billeddannelse af bakterieceller for nøjagtige cellulære repræsentationer og præcis 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 ...