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 chilled 10x PBS (pH 7.0 -&#160;7.3) to 400 &#181;L of chilled collagen stock solution. Mix the solution by gently tiling the tube. 
	Note: All steps in Part 1 should be done under a cell culture hood.</li>
	<li>Add 8&#160;&#181;L of 0.1 M NaOH into 500 &#181;L of the collagen solution from 1.1. to adjust the pH to 7.2-7.6. Use pH test strips (pH range: 6 - 10) to determine the pH value of the mixture. 
	Note: Volumes may vary for different batches of collagen. NaOH solution has to be mixed slowly to avoid air bubbles. The mixture should be kept on ice to avoid collagen gelation.</li>
	<li>Add 2&#160;&#181;L of sterile ddH2O to make the final volume to 500 &#181;L. Mix well and store this collagen solution (8.32 mg/mL) on ice or at 4 &#176;C until further use. 
	Note: Under this condition, neutralized collagen can be used for 24 h. Aliquots are not recommended to avoid air bubbles.</li>
</ol>
2. Sample Preparation for Light-sheet Fluorescence Microscopy Using Capillaries
<ol>
	<li>Fluorescently label the live cells of interest with the desired primary fluorescent dyes15 or fluorescent proteins11 as described previously.</li>
	<li>Transfer 1 &#215; 106 of cells into a sterile 1.5 mL tube under a cell culture hood. Centrifuge the tube at 200 x g for 8 min. Discard the supernatant and resuspend the pellet in 200 &#181;L of culture medium. 
	Note: The cell density of 5 &#215; 106 cells/mL is recommended for visualization of human immune cell migration, especially for cytotoxic T lymphocytes (CTL) and natural killer (NK) cells.</li>
	<li>Add 85.9 &#181;L of neutralized collagen solution from 1.3. into the cell suspension from 2.2. and mix properly to reach a collagen concentration of 2.5 mg/mL. Leave the cell/collagen mix on ice in the hood. 
	Note: Suppose the desired concentration of collagen is N mg/mL, the volume of neutralized collagen solution (for 200 &#181;L cell suspension) = 200 &#215; N/(8.32 - N)</li>
	<li>Next, put the matching plunger into the capillary (inner diameter ~1 mm) until the plunger is 1 mm out of the capillary. Wet the plunger by dipping into culture medium&#160;(Figure 1A). 
	Note: This step could help to prevent air bubbles when the capillary is dipped into the cell/collagen mix. The capillary and the plunger do not have to be sterile.</li>
	<li>Dip the capillary into the cell/collagen mix from 2.3. Slowly pull the plunger back for 10 -&#160;20 mm (Figure 1B). Wipe the outer wall of the capillary with a paper towel moistened with 70% ethanol spray to remove the remaining collagen solution.</li>
	<li>Mount the capillary&#160;with modeling clay on the inner wall of a 5 mL tube and push the cell/collagen mix to the edge of the capillary&#160;(Figure 1C).</li>
	<li>Keep the capillary at 37 &#176;C with 5% CO2 for 1 h for collagen polymerization.</li>
	<li>Add the culture medium (around 1 - 2 mL) to a 5 mL tube. Carefully press the polymerized collagen rod out into the medium to around 3/4 of the collagen hanging in the medium (Figure 1D).</li>
	<li>Keep the capillary, like this, at 37 &#176;C with 5% CO2 for another 30 min to equilibrate the collagen rod with the medium. 
	Note: Afterwards, the collagen rod can be pulled back into the capillary and cultured further before further use.</li>
</ol>
3. Image Acquisition using Light-sheet microscopy
<ol>
	<li>Assembly the sample chamber according to the manufacturer&#8217;s instruction.</li>
	<li>Turn on the incubation and the microscope to heat the sample chamber to 37 &#176;C (for live cell imaging only).</li>
	<li>Place the capillary in the sample chamber and locate the sample to&#160;find the area of interest for image acquisition.</li>
	<li>Activate the corresponding laser(s). Set the following settings: laser power, exposure time, step-size of z-stack, start and end position of z-stack, and the time interval for live cell imaging. 
	Note: For example, the sample in Figure 2 was imaged every 40 s for 6 h at 37 &#176;C with a step-size of 1 &#181;m (total thickness: 538 &#181;m). The laser power was 1% with an exposure time of 30 ms. The pixel size at x-y direction is 0.23 &#181;m.</li>
	<li>Start the image acquisition.</li>
</ol>
4. Automated Tracking Analysis
<ol>
	<li>Open the file converter, click Add Files to choose the imaging file(s) to be converted into the software file format (*.ims). Click Browse and select a folder to save the converted file(s). Click Start All.</li>
	<li>Open the analysis software. Click Surpass. Go to File and click Open, then choose the imaging file to be analyzed. 
	Note: If the file size is big this step may take time. Files larger than 1 Terabyte (TB) are not recommended for a single experiment as the process such big files are highly computational capacity demanding.</li>
	<li>Click Add New Spots. Check the box Process Entire Image Finally. Click Next.</li>
	<li>Enter the coordinates for x and y (in pixel) to define the region of interest. Enter the number of frames (time and z-position) to analyze. Click Next.</li>
	<li>Select the target channel, which contains the objects to be tracked, in the Dropdown list of Source Channel. Enter Estimated xy Diameter (in &#181;m). Click Next. 
	Note: Estimated xy diameter is the average diameter in the x-y dimension of objects to be tracked.</li>
	<li>Click Quality. Set a threshold, in which most of the cells (objects) should be included. Click Next.&#160;</li>
	<li>Choose the desired algorithm (Autoregressive Motion is recommended). Enter the max distance (20 &#181;m is recommended) and the max gap size (3 or 2 is recommended). Click Process Entire Image finally. 
	Note: Max distance and Max gap size are two thresholds to break tracks. More specifically, in two continuous frames when the distance between the same object exceeds the Max distance, this object in the later frame will be considered as a new object. Sometimes during acquisition, the same object may disappear for a few frames and show up again. In this case, only when this object reappears within the Max gap size, it will be considered as the same object.</li>
	<li>Click Filter Type and choose the option to exclude undesired tracks. 
	Note: This step is optional.</li>
	<li>Click Next and then click Finish. 
	Note: This step can take hours up to days depending on computing performance.</li>
	<li>Click Edit Tracks and choose Correct Drift. Select the appropriate algorithm (Translational Drift is recommended). Select the desired Result Dataset Size (New Size Equal to Current Size is recommended). Click OK. 
	Note: This step is only required when the collagen drifted during image acquisition.</li>
	<li>Click Statistics and choose Configure List of Visible Statistics Values. Check the options of interest to be exported (e.g. coordinates, speed and so on). Click OK.</li>
	<li>Click Export All Statistics to File and enter the file name.</li>
</ol>
5. Fixation and Immunofluorescence Staining of Cells in Collagen Matrices
<ol>
	<li>Transfer 1,000 &#181;L of 4% paraformaldehyde (PFA, in PBS) into a 5 mL tube under a chemical hood. 
	Note: PFA should be balanced to room temperature.</li>
	<li>Dip the capillary with polymerized collagen from 2.9. into PFA solution (for ~ 5 mm) and mount the capillary on the inner wall of the 5 mL tube with modeling clay (as shown in Figure 1C).</li>
	<li>Press the plunger gently until half of the collagen rod is hanging in the PFA solution (Figure 1D). Keep the tube at room temperature for 20&#160;min.</li>
	<li>Pull back the plunger to get the collagen rod inside the capillary. Take out the capillary and discard PFA.</li>
	<li>Mount the capillary into a fresh tube and add 1 mL of PBS. Make sure that the capillary is immersed in the PBS.</li>
	<li>Press the plunger gently until half of the collagen rod is hanging in the solution. Mount the capillary on the inner wall with modeling clay.</li>
	<li>Keep the tube at room temperature for 5 min.</li>
	<li>Repeat 5.4. - 5.7 for another 2 times.</li>
	<li>Pull back the plunger to get the collagen rod inside the capillary. Discard PBS. Transfer 1 -&#160;2 mL of blocking/permeabilization buffer (PBS + 1-% BSA + 0.1% non-ionic surfactant) into the tube and repeat 5.6. 
	Note: The BSA (1%) can be replaced by&#160;5% serum of the animal the secondary Ab was raised in.</li>
	<li>Keep the tube at room temperature for 30 - 60 min.</li>
	<li>Pull back the plunger to get the collagen rod inside the capillary. Discard the permeabilization buffer. Transfer 200 - 500 &#181;L of the primary antibody in blocking/permeabilization buffer and expels the rod into the solution.</li>
	<li>Keep the tube at room temperature for 1 h.</li>
	<li>Wash the collagen rod 3 times with PBST (PBS + 0.1-% non-ionic surfactant) as described in 5.4. -&#160;5.8.</li>
	<li>Incubate the rod in&#160;secondary antibody in blocking/permeabilization buffer for 1 h at room temperature. Keep it from light.</li>
	<li>Wash the collagen rod 3 times with PBS as described in 5.4. -&#160;5.8.</li>
	<li>Pull back the plunger to get the collagen rod inside the capillary. Keep the samples in PBS until imaging.</li>
	<li>Scan the samples as described in 3.</li>
</ol>

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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. 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The authors declare no financial or commercial conflict of interest.

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 ...

light-sheet-microscopy-for-three-dimensional-visualization-human

Research

JoVE Journal

Immunology and Infection

8.0K Views.  Saarland University. 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.

Video: Light-sheet Microscopy for Three-dimensional Visualization of Human Immune 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 cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

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

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

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

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

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

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

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

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Visualization of the Immunological Synapse by Dual Color Time-gated Stimulated 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 actin reorganization is critical to the function of NK cells and the formation of the IS. Imaging of F-actin at the synapse has traditionally utilized confocal microscopy, however the diffraction limit of light restricts resolution of fluorescence microscopy, including confocal, to approximately 200 nm. Recent advances in imaging technology have enabled the development of subdiffraction limited super-resolution imaging. In order to visualize F-actin architecture at the IS we recapitulate the NK cell cytotoxic synapse by adhering NK cells to activating receptor on glass. We then image proteins of interest using two-color stimulated emission depletion microscopy (STED). This results in &#60;80 nm resolution at the synapse. Herein we describe the steps of sample preparation and the acquisition of images using dual color STED nanoscopy to visualize F-actin at the NK IS. We also illustrate optimization of sample acquisition using Leica SP8 software and time-gated STED. Finally, we utilize Huygens software for post-processing deconvolution of images.

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

Here we illustrate the protocol for imaging by two-color STED nanoscopy the cytotoxic immune synapse of NK cells recapitulated on glass. Using this method we obtain sub-100 nm resolution of synapse proteins and the cytoskeleton.

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

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

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

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease and treatment. It has the potential to predict therapeutic efficacy and identify novel therapeutic targets. Whole blood staining utilizes unmanipulated blood, which minimizes artifacts that can occur during sample preparation. However, whole blood staining must also be done on freshly collected blood to ensure the integrity of the sample. Additionally, it is best to prepare antibody cocktails on the same day to avoid potential instability of tandem-dyes and prevent reagent interaction between brilliant violet dyes. Therefore, whole blood staining requires careful standardization to control for intra and inter-experimental variability.
Here, we report deployment of an automated liquid handler equipped with a two-dimensional (2D) barcode reader into a standard process of making antibody cocktails for flow cytometry. Antibodies were transferred into 2D barcoded tubes arranged in a 96 well format and their contents compiled in a database. The liquid handler could then locate the source antibody vials by referencing antibody names within the database. Our method eliminated tedious coordination for positioning of source antibody tubes. It provided versatility allowing the user to easily change any number of details in the antibody dispensing process such as specific antibody to use, volume, and destination by modifying the database without rewriting the scripting in the software method for each assay.
A proof of concept experiment achieved outstanding inter and intra- assay precision, demonstrated by replicate preparation of an 11-color, 17-antibody flow cytometry assay. These methodologies increased overall throughput for flow cytometry assays and facilitated daily preparation of the complex antibody cocktails required for the detailed phenotypic characterization of freshly collected anticoagulated peripheral blood.

Whole blood Immunophenotyping is indispensable for monitoring the human immune system. However, the number of reagents required and the instability of specific fluorochromes in premixed cocktails necessitates daily reagent preparation. Here we show that semi-automated preparation of staining antibody cocktail helps establish reliable immunophenotyping by minimizing variability in reagent dispensing.

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

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling and cellular processes that control efferocytosis. This quantification can be difficult to perform as apoptotic cells are often efferocytosed piecemeal, thus necessitating methods which can accurately delineate between the efferocytosed portion of an apoptotic target versus residual unengulfed cellular fragments. The approach outlined herein utilizes dual-labeling approaches to accurately quantify the dynamics of efferocytosis and efferocytic capacity of efferocytes such as macrophages. The cytosol of the apoptotic cell is labeled with a cell-tracking dye to enable monitoring of all apoptotic cell-derived materials, while surface biotinylation of the apoptotic cell allows for differentiation between internalized and non-internalized apoptotic cell fractions. The efferocytic capacity of efferocytes is determined by taking fluorescent images of live or fixed cells and quantifying the amount of bound versus internalized targets, as differentiated by streptavidin staining. This approach offers several advantages over methods such as flow cytometry, namely the accurate delineation of non-efferocytosed versus efferocytosed apoptotic cell fractions, the ability to measure efferocytic dynamics by live-cell microscopy, and the capacity to perform studies of cellular signaling in cells expressing fluorescently-labeled transgenes. Combined, the methods outlined in this protocol serve as the basis for a flexible experimental approach that can be used to accurately quantify efferocytic activity and interrogate cellular signaling pathways active during efferocytosis.

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

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

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 affected by cell shape. Bacterial cells are three-dimensional (3D) objects, although they are rarely treated as such. Most microscopy techniques result in two-dimensional (2D) images leading to the loss of data pertaining to the actual 3D cell shape and localization of proteins. Certain shape parameters, such as Gaussian curvature (the product of the two principal curvatures), can only be measured in 3D because 2D images do not measure both principal curvatures. Additionally, not all cells lie flat when mounting and 2D imaging of curved cells may not accurately represent the shapes of these cells. Accurately measuring protein localization in 3D can help determine the spatial regulation and function of proteins. A forward convolution technique has been developed that uses the blurring function of the microscope to reconstruct 3D cell shapes and to accurately localize proteins. Here, a protocol for preparing and mounting samples for live cell imaging of bacteria in 3D both to reconstruct an accurate cell shape and to localize proteins is described. The method is based on simple sample preparation, fluorescent image acquisition, and MATLAB-based image processing. Many high-quality fluorescent microscopes can be simply modified to take these measurements. These cell reconstructions are computationally intensive and access to high-throughput computational resources is recommended, although not necessary. This method has been successfully applied to multiple bacterial species and mutants, fluorescent imaging modalities, and microscope manufacturers.

This protocol explains how to prepare and mount bacterial samples for live three-dimensional imaging and how to reconstruct the three-dimensional shape of E. coli from those images.

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 ...