We thank the Institute for Clinical Hemostaseology and Transfusion Medicine for providing donor blood; Carmen H&#228;ssig and Cora Hoxha for excellent technical help. We thank Jens Rettig (Saarland University) for the modified pMAX vector, Roland Wedlich-S&#246;ldner (University of Muenster) for the original LifeAct-Ruby construct, and Christian Junker (Saarland University) for generating the LifeAct-mEGFP construct. This project was funded by Sonderforschungsbereich 1027 (project A2 to B.Q.) and 894 (project A1 to M.H.). The light-sheet microscope was funded by DFG (GZ: INST 256/4 19-1 FUGG).

Research carried out for this study with the human material (leukocyte reduction system chambers from human blood donors) is authorized by the local ethics committee (declaration from 16.4.2015 (84/15; Prof. Dr. Rettig-St&#252;rmer)) and follows the corresponding guidelines.
1. Preparation of Neutralized Collagen Solution (500 &#181;L)
<ol>
	<li>Transfer 400 &#181;L of chilled collagen stock solution (10.4 mg/mL) to a sterile 1.5 mL tube under the cell culture hood. Slowly add 50 &#181;L of ...

<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. F., Bartumeus, F., Gerard, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+migration,+search+strategies+and+mechanisms.">T cell migration, search strategies and mechanisms.</a> Nat Rev Immunol. 16 (3), 193-201 (2016).</li><li>Entschladen, F., 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=Analysis+methods+of+human+cell+migration.">Analysis methods of human cell migration.</a> Exp Cell Res. 307 (2), 418-426 (2005).</li><li>Meredith, J. E., Fazeli, B., Schwartz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extracellular+matrix+as+a+cell+survival+factor.">The extracellular matrix as a cell survival factor.</a> Mol Biol Cell. 4 (9), 953-961 (1993).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+cell+migration:+a+multiscale+tuning+model.">Plasticity of cell migration: a multiscale tuning model.</a> J Cell Biol. 188 (1), 11-19 (2010).</li><li>Ridley, A. 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=Cell+migration:+integrating+signals+from+front+to+back.">Cell migration: integrating signals from front to back.</a> Science. 302 (5651), 1704-1709 (2003).</li><li>Shamir, E. R., Ewald, A. 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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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Most in vitro assays are carried out on a 2D surface, for example in cell-culture plates, Petri-dishes or on coverslips, whereas in vivo cells, especially immune cells, experience mostly a 3D microenvironment. Emerging evidence shows that migration patterns of immune cells differ between 2D and 3D scenarios17. Moreover, the expression profiles of tumor cells are also different in 2D- and 3D-cultured tissues18,19,</su...

Most knowledge about migrating cells comes from 2D experiments1,2,3, which are normally conducted in a glass or plastic surface of a culture/imaging dish. However, a physiological scenario requires, in most cases, a 3D microenvironment, in which the extracellular matrix (ECM) plays a decisive role. ECM not only provides the 3D structure essential to maintain proper cell morphology but also offers survival signals or directional cues for an optimal functioning of many cells4,5 . Therefore, a 3D environment is required to better identify cellular functions and behavior in an environment better reflecting the physiological context.
In the human body, most cells especially immune cells, exert their functions under a 3D scenario. For example, activated T cells patrol tissues searching for target cells, na&#239;ve T cells migrate through lymph nodes in search for their cognate antigen-presenting cells during which the migration mode and machinery are adapted to the corresponding extracellular environment3,6,7. The 3D collagen gel has been widely used as a well-established and well-characterized 3D cell culture system8,9,10. Our previous work shows that primary human lymphocytes are highly mobile and migrate at an average speed of around 4.8 &#181;m/min in a 0.25 % collagen-based matrix11. Rearrangement of cytoskeleton plays a key role in the cell migration12. Accumulating evidence shows that lymphocytes do not apply only a single mode of migration yet can switch between certain migration behavior depending on the location, microenvironment, cytokines, chemotactic gradients, and extracellular signals which tune the migratory behavior in different ways 3.
To reliably analyze immune cell functions and behavior, for example, migration, protrusion formation or vesicular transportation, it is of great advantage to be able to acquire images in relatively large 3D volumes in a fast and reliable manner. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) offers a satisfactory solution13,14. During imaging acquisition, a thin static light sheet is generated to illuminate the sample. In this way, on the focus plane, a large area can be illuminated simultaneously without affecting the off-plane cells. This feature enables a high acquisition speed with a drastically reduced bleaching and photocytotoxicity. In this paper, we describe how to visualize primary human immune cells using light-sheet microscopy and how to analyze the migration in a 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, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Protrusion formation during T cell migration is a highly dynamic process, which is actin dependent. To visualize protrusion formation of primary human CTL, we transiently transfected a mEGFP fused protein to label the actin cytoskeleton in CTL as described before11. One day after transfection, the cells were embedded in the collagen matrix. Image stacks were acquired every 40 s with a step-size of 1 &#181;m at 37 &#176;C using light-sheet microscopy. As shown in <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

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 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 ...

This method can help answer key questions in the immunology field such as how exactly immune cells behave in a physiologically relevant context like how to search target cells efficiently in a three-dimensional environment. The main advantage of this method is to generate a very thin sheet of light to illuminate only the focal plane without affecting the off plane cells. This enables a very fast acquisition speed with a pronounced reduction of bleaching and photocytotoxicity.Demonstrating this procedure will be Dr.Renping Zhao, a post-doc from my lab. To begin, under a cell culture hood, transfer 400 microliters of chilled collagen stock solution to a sterile 1.5 milliliter tube. Slowly add 50 microliters of chilled 10X PBS.Then mix the solution by gently tilting the tube. Add 48 microliters of 0.1 molar sodium hydroxide to the collagen solution to adjust the pH to 7.2 to 7.6. Use pH test strips to determine the pH value of the mixture.Add two microliters of sterile distilled deionized water to bring the final volume to 500 microliters. Mix well and store the collagen solution on ice or at four degrees Celsius until further use. After fluorescently labeling live cells according to the text protocol, under the cell culture hood, transfer one times 10 to the sixth cells into a sterile 1.5 milliliter tube.Centrifuge the tube at 200 times g for eight minutes. Then discard the supernatant and use 200 microliters of culture medium to resuspend the pellet. Add 85.9 microliters of the neutralized collagen solution to the cell suspension and mix properly to reach a collagen concentration of 2.5 milligrams per milliliter.Leave the cell collagen mix on ice in the hood. Next, insert a plunger into the matching capillary until the plunger is sticking one millimeter out of the capillary. Then wet the plunger by dipping into culture medium.Skipping this step can result in undesirable introduction of air bubbles between the plunger and the collagen matrix. Dip the capillary into the cell collagen mixture and slowly pull the plunger back 10 to 20 millimeters. Then with a spray bottle of 70%ethanol, moisten a paper towel and use it to wipe the outer wall of the capillary to remove the remaining collagen solution.Now use modeling clay to mount the capillary to the inner wall of a five milliliter tube. Push the cell collagen mix to the edge of the capillary. Then incubate the tube with the capillary at 37 degrees Celsius and 5%CO2 for one hour to polymerize the collagen.Following the incubation, add one to two milliliters of culture medium to the tube. Then carefully expel the polymerized collagen rod out into the medium until approximately half of the collagen is hanging in the medium. Incubate the capillary for another 30 minutes.To carry out light-sheet microscopy, assemble the sample chamber according to the manufacturer's instructions. After turning on the microscope and incubator if doing live imaging, place the capillary in the sample chamber, locate the sample, and find the area of interest for image acquisition. Activate the corresponding lasers.Then set the laser power and exposure time. Also set the step size of the Z stack, the start and end positions of the Z stack, and the time interval for live cell imaging. Then start the image acquisition.Transfer one milliliter of 4%paraformaldehyde in PBS into a five milliliter tube under a chemical hood. Then dip the capillary with the polymerized collagen into the paraformaldehyde solution and use modeling clay to mount the capillary on the inner wall of the tube. Gently press the plunger until half of the collagen rod is hanging in the paraformaldehyde solution.Then pull back the plunger to take the collagen rod back up into the capillary. Remove the capillary from the tube and discard the paraformaldehyde. Mount the capillary into a fresh tube and add one milliliter of PBS.Ensure that the capillary is immersed in the PBS. Gently press the plunger until half of the collagen rod is hanging in the solution and incubate for five minutes. Pull back the plunger to take up the collagen rod in the capillary.Then replace the PBS with fresh PBS and expel the collagen rod into the solution. Following the third wash, add one to two milliliters of blocking permeabilization buffer into the tube. Expel the collagen rod into the solution and incubate the tube at room temperature for 30 to 60 minutes.Replace the blocking permeabilization buffer with 200 to 500 microliters of primary antibodies in blocking permeabilization buffer and incubate the submerged collagen rod in the solution for one hour. After using PBST to wash the collagen rod three times, incubate the rod in secondary antibodies in blocking permeabilization buffer at room temperature for one hour. Following three more washes in PBS, pull the collagen rod back into the capillary and keep the sample in PBS until imaging.As seen in this movie, during migration CTL cells transfected with an EGFP actin fusion protein formed lemon-shaped major protrusions, fringed by fine spindle-like structures. The trajectories of CTLs are illustrated here with velocity and persistence or the displacement divided by the total track length as the measured parameters. The velocities range from 0.01 to 0.19 microns per second with an almost 20-fold difference and the persistence ranges from zero to 0.7.This figure shows fixed CTL cells in 3D collagen gel stained for endogenous perforin-1 and actin. The sample was illuminated from a single side or from both sides. From different Z positions, the cells are evenly stained indicating good penetration of antibodies into the collagen gels.Fixed CTL exhibited the same morphology as live cells indicating that the morphology is well-maintained with this protocol. While attempting this procedure, it's very important to remember to avoid air bubbles and to adjust pH value properly. Following this procedure, other methods like live cell staining with particular surface molecules can be performed to answer additional questions like how to correlate cell behavior with differentiation or with different cell subtypes or to investigate cell-cell interaction and cell fate.After its development, this technique paved the way for researchers in the field of cell biology, immunology, and cancer research to explore cell behavior, cell fate, differentiation, and cell interaction in the most physiologically relevant system in vitro. After watching this video, you should have a good understanding of how to prepare samples with cells embedded in a three-dimensional collagen matrix, how to visualize samples using light-sheet microscopy, either live or fixed, and how to track cell migration in 3D.

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Research

JoVE Journal

Immunology and Infection

8.0K ビュー.  Saarland University. This method can help answer key questions in the immunology field such as how exactly immune cells behave in a physiologically relevant context like how to search target cells efficiently in a three-dimensional environment. The main advantage of this method is to generate a very thin sheet of light to illuminate only the focal plane without affecting the off plane cells. This enables a very fast acquisition speed with a pronounced reduction of bleaching and photocytotoxicity.Demonstrat...