This work was supported by grants from the &#34;Deutsche Forschungsgemeinschaft&#34; SFB 854/TP11 to K.-D.F.

All experiments involving the use of animals have been previously approved by the Landesverwaltungsamt Halle (Saxony-Anhalt), Germany, in accordance with all guidelines of the medical faculty of the OVGU University of Magdeburg.
1. MZB Cell Purification
<ol>
	<li>Isolate leukocytes.
	<ol>
		<li>Sacrifice an 8&#160;to 16 week-old mouse and remove the spleen16. Dissociate the spleen in 5 mL of ice-cold cell buffer [phosphate-buffered saline (PBS) + 0.5% fatty acid-free bovine serum albumin (BSA)], by either using a tissue dissociator or gently mashing the spleen through a 70 &#181;m cell strainer mesh into a well on a 6-well plate with the plunger from a 5 mL syringe.</li>
		<li>Transfer the cells in cell buffer into a 15 mL conical tube. Collect additional cells by adding another 5 mL of ice-cold cell buffer to the tissue dissociator tube or the nylon mesh in the well. Transfer the additional 5 mL of cell buffer plus cells to the conical tube. Centrifuge at 300 x g for 10 min at room temperature (RT, 20&#160;&#176;C) and discard the supernatant.</li>
	</ol>
	</li>
	<li>Perform red blood cell lysis.
	<ol>
		<li>Re-suspend the cell pellet in 1 mL of red blood cell (RBC) lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) that is pre-warmed to RT. Add an additional 1.5 mL of RBC lysis buffer and swirl to mix. Incubate at RT (20&#160;&#176;C) for a total of 2 min, including the time required to re-suspend the pellet.</li>
		<li>Add 7.5 mL of ice-cold cell buffer to the cell suspension to stop the lysis. Centrifuge at 300 x g and 4&#160;&#176;C for 10 min and discard the supernatant.</li>
		<li>Re-suspend the cell pellet in 1 mL of cell buffer and add an additional 9 mL of cell buffer. Pipette the 10 mL cell suspension through a 30 &#181;m pre-separation filter into a new 15 mL conical tube to remove cell debris.</li>
		<li>Centrifuge cells at 300 x g and 4&#160;&#176;C for 10 min and discard the supernatant. Re-suspend the cell pellet in 500 &#181;L of cell buffer. Keep cells cold at all subsequent steps of the procedure until incubation in the flow chamber slides.</li>
	</ol>
	</li>
	<li>Purify MZBs.
	<ol>
		<li>Add 50 &#181;L of biotin-coupled antibodies from a commercially available kit against all undesired cells, including CD43 (on non-B cells), CD4 (on T cells), CD93 (on immature B cells), and Ter119 (on erythrocytes), to the cell suspension in a conical tube. Shake or flick the tube gently to mix, but do not vortex the cells. Lay the conical tube on a bed of ice at an almost-flat angle and rock at 25 rpm for 15 min.</li>
		<li>Add magnetic beads coupled to anti-biotin antibodies in a total volume of 100 &#181;L to the cell suspension. Continue to rock the cell suspension at 25 rpm on ice for 20 min. Wash the cells by adding 5 mL of cell buffer, centrifuging at 300 x g and 4&#160;&#176;C for 10 min, and discarding the supernatant. Re-suspend cells in 1 mL of cell buffer.</li>
		<li>Deplete the cell suspension of the magnetic-bead-coupled cells by retaining them on a magnetic column. Discard labeled cells (all non-B cells and immature B cells). Collect the non-labeled cells (mature B cells) in a 15 mL conical tube that has been pre-coated with a brief application of 5 mL of cell buffer, in order to prevent non-specific adhesion by MZBs.</li>
		<li>Wash the cell suspension with 5 mL of cell buffer. Centrifuge at 300 x g and 4&#160;&#176;C for 10 min and discard the supernatant. Re-suspend cells in 90 &#181;L of cell buffer and add 10 &#181;L of anti-CD23-coupled beads. Rock cell suspension gently on ice for 20 min at 25 rpm.</li>
		<li>Wash the cell suspension with 5 mL of cell buffer. Centrifuge at 300 x g and 4&#160;&#176;C for 10 min and discard the supernatant. Re-suspend cells in 1 mL of cell buffer.</li>
		<li>Deplete the cell suspension of the magnetic-bead-coupled cells by retaining them on a magnetic column. Discard the labeled cells (follicular B cells). Collect the non-labeled cells (MZBs), in a 15 mL conical tube that has been pre-coated with a brief application of 5 mL of cell buffer. Remove 50 &#181;L of the cells for counting and checking purity.</li>
	</ol>
	</li>
	<li>Stain the sorted MZBs to check for purity.
	<ol>
		<li>Add 10% volume (5 &#181;L) of Fc block to 10,000 cells in 50 &#181;L and incubate at 4&#160;&#176;C for 5 min. Add 50 &#181;L of cell buffer containing 0.3 &#181;L each of B220-FITC (0.15 &#181;g), CD21-PE (0.06 &#181;g), and CD23-APC (0.06 &#181;g) in a total volume of 105 &#181;L (each antibody diluted 1:350), or any other combination of fluorophores for distinguishing between follicular B cells and MZBs. Stain the cells at 4&#160;&#176;C in the dark for 15 min.</li>
		<li>Wash the stained cells once with 1 mL of cell buffer. Centrifuge at 300 x g and 4&#160;&#176;C for 5 min and discard the supernatant. Resuspend in 300 &#181;L of cell buffer and acquire samples using a flow cytometer and standard methods. Gate on live lymphocytes, then on B220+ cells, and finally on CD23low CD21high cells (Figure 1B). 
		NOTE: The procedure typically yields 500,000 MZBs per spleen from a C57B/6 mouse.</li>
	</ol>
	</li>
	<li>Prepare MZBs for flow migration.
	<ol>
		<li>Wash purified MZBs once in 1&#8211;2 mL of cold migration buffer [Hanks&#8217; balanced salt solution (HBSS) containing cations, 10 mM Hepes, 0.2% fatty acid-free BSA] to ensure that the cation-containing migration buffer is not diluted by residual cation-free PBS buffer. Centrifuge at 300 x g and 4&#160;&#176;C for 10 min and discard the supernatant. Re-suspend MZBs in cold migration buffer to a concentration of 50,000 MZBs per 30 &#181;L (equivalent to the amount loaded in one well of a flow chamber slide). 
		NOTE: The freshly isolated MZBs should be used as soon as possible. However, they are still capable of migrating for up to 8 h or more after the start of the experiment for as long as they are kept on ice. Optimal temperatures should be tested for other cell types. For example, it may be better to keep cultured T cells at 37&#160;&#176;C until the start of migration.</li>
	</ol>
	</li>
</ol>
2. Flow Experiment
<ol>
	<li>Coat slides with integrin ligand.
	<ol>
		<li>On the day before the experiment, thaw an aliquot of ICAM-1 (400 &#181;g/mL). Dilute ICAM-1 by a factor of 1:80 in PBS to reach a final concentration of 5 &#181;g/mL. Add 30 &#181;L of ICAM-1 to one chamber on a flow slide for every migration movie required. Incubate slide in a humid chamber at 4&#160;&#176;C overnight. 
		NOTE: Each slide contains 6 chambers and up to 8 movies in one session can be easily recorded.</li>
	</ol>
	</li>
	<li>Block the coated wells.
	<ol>
		<li>On the day of the experiment, wash the chamber by adding 100 &#181;L of PBS to one well and withdrawing 100 &#181;L from the other well of the chamber. Add 100 &#181;L of blocking buffer (2% fatty acid-free BSA in PBS) to a chamber and incubate the slide in a humid chamber at RT for 1.5 h.</li>
		<li>Wash the chamber by adding 100 &#181;L of PBS to one well, withdrawing it from the other well, then adding 100 &#181;L of migration buffer to the chamber. Store the slides in a humid chamber at RT until the experiment.</li>
	</ol>
	</li>
	<li>Prepare the fluidics unit.
	<ol>
		<li>Plug in a fluid pump, attach the fluidics unit, and turn on the pump software. Wash the tubing once with 5&#8211;10 mL of migration buffer, then add 11.7 mL of migration buffer equally to both reservoirs and remove the air bubbles.</li>
		<li>Clamp the tubing about 10 cm from the connector piece. Place the fluidic unit in the heated incubation chamber on the microscope.</li>
		<li>Set the slide choice to &#8220;&#181;-slide VI 0.4&#8221; and the tubing to &#8220;white&#8221; in the software. Set the desired shear stress (e.g., 4 dyn cm-2). Set the time of imaging to 30 min.</li>
		<li>Set the tubing calibration factor by determining the flow rate through the tubing in mL/min. Measure the time required for 2 mL of the buffer to flow from one reservoir of the other and divide by 2. Make at least 4 measurements for accuracy. Use the actual flow rate to set the desired flow rate and shear stress by entering the values into the pump software.</li>
	</ol>
	</li>
	<li>Prepare the imaging system.
	<ol>
		<li>Pre-warm the microscope incubation chamber, fluidics unit, and migration buffer aliquots to 37&#160;&#176;C (Figure 2B). Test the fluidic pump by ensuring that the migration buffer flows back and forth in the reservoirs and that there are no air bubbles in the system.</li>
	</ol>
	</li>
	<li>Load the cells on the slide.
	<ol>
		<li>Clean the bottom of the slide with an ethanol-dampened tissue. Add 30 &#181;L of the cell suspension into one well of the chamber and withdraw 30 &#181;L from the other well. Incubate the slide with a cover on to prevent evaporation at 37&#160;&#176;C for 30 min to allow the cells to attach.</li>
		<li>Slowly add the pre-warmed migration buffer to each well of the flow chamber slide until a positive meniscus rises out of the well and remove any air bubbles with the pipette tip.</li>
	</ol>
	</li>
	<li>Attach the slide to the fluidics unit.
	<ol>
		<li>Clamp the slide to the microscope stage. Remove the unmarked side of the fluidic tubing from the connector piece and fill up the end with migration buffer until a positive meniscus without air bubbles rises out of the end.</li>
		<li>Flip the end of the tubing over and insert it into the top well of the flow chamber, mopping up spilled buffer with a tissue. While keeping the tubing clamped, repeat the procedure for the marked end of the tubing.</li>
	</ol>
	</li>
	<li>Image the cells.
	<ol>
		<li>Switch the imaging system to &#8220;Live&#8221; to set the focus on the cells. Select a field of view that is in the middle of the slide. De-focus the cells slightly to enhance the black outline of the cell and produce a white interior, which can then be thresholded to a round black cell shape on a white background for use with an automated tracking program.</li>
		<li>Start the imaging sequence program and record 1 image every 5 s for 30 min using the 10x dry objective. Remove the clamp from the tubing and turn the pump on, then non-adherent cells will wash off and adherent cells will begin to migrate. Image for 30 min or longer using a 10x objective or higher, as desired. Label and save the movie.</li>
	</ol>
	</li>
	<li>Detach the fluidic unit from the slide.
	<ol>
		<li>At the end of the imaging program, reset the pump for the next experiment: re-attach the clamp to the tubing, remove the tubing from the slide, re-attach the connector piece, open the clamp, and use the manual control of the pump to push several mL of buffer from one reservoir to the other to remove the air bubbles. Re-attach the clamp and lay the clamped tubing on the microscope stage for the next movie. 
		NOTE: The protocol can be paused indefinitely here. If using an inhibitor or modifier of cell migration, depending on its mode of action, add it to either the cell suspension before settling in the migration chamber or to the migration buffer in the fluidic unit.</li>
	</ol>
	</li>
</ol>
3. Migration Track Analysis
NOTE: Cells can be tracked automatically using the MTrack2 plugin or by hand using the Manual Tracking plugin17. Automatic tracking works well with MZBs because these cells are mainly round and remain this way while migrating, making it easy to threshold the image of the cells to black objects on a white background. Automatic tracking is more difficult if other cell types are used, such as cultured, activated CD8+ T cells, because these cells stretch out during migration and become somewhat transparent, making it difficult to define the edges. In this case, either (1) the cells can be stained with an intra-vital fluorescent dye to produce images that can be thresholded to show black objects on a white background, or (2) other programs to outline the cells such as image segmentation and/or edge detection can be used. Manual tracking is a useful option when producing a high-contrast image of cell outlines is not possible.
<ol>
	<li>Perform manual tracking using the plugin.
	<ol>
		<li>Install the &#8220;Manual Tracking&#8221; plugin in ImageJ and open the movie in ImageJ. Under the &#8220;Manual Tracking&#8221; menu, select &#8220;add new track&#8221;. Click the center of a cell as the movie advances automatically frame-by-frame. When the cell has been selected in every frame, select &#8220;add new track&#8221; to start following a new cell.</li>
		<li>After the tracks have been recorded, copy the output results into a data spreadsheet. Change the format for decimal display for all spreadsheet cells to zero places after the decimal. Save the file as a &#8220;tab stop-separated&#8221; .txt file for subsequent analysis. 
		NOTE: Typically, 50&#8211;100 cells are tracked, which takes about 1 h to complete for a 20 min movie of about 240 frames.</li>
	</ol>
	</li>
	<li>Perform automatic tracking using the MTrack2 plugin. Download and install the MTrack2 kt plugin according to instructions in the file (see Supplemental Coding File).
	<ol>
		<li>Threshold the images from the cell migration movie. Open the image stack in ImageJ. Process the images in ImageJ to convert the video from color to high-contrast images showing the cells as black objects on a white background (Figure 3A).
		<ol>
			<li>Sharpen the images twice, despeckle the images, and convert the images to 8 bit. Select the &#8220;Image | adjust brightness/contrast&#8221; sub-menu and put the contrast slider at maximum contrast, then the cells will appear as white objects on a black background. Select the &#8220;Image | adjust threshold&#8221; sub-menu to ensure that the cells appear as black objects on a white background.</li>
		</ol>
		</li>
		<li>Run the automatic cell tracking plugin &#8220;MTrack2 kt&#8221; (see Supplemental Coding File). Set the following parameters on the options screen.
		<ol>
			<li>Set the particle size minimum at 1 pixel and particle size maximum at 30 pixels for MZBs.</li>
			<li>For velocity (maximum distance between 2 particles on successive frames of the movie), if the cell density on the slide is high, set this value low to avoid having the track &#8220;jump&#8221; from one cell to another. For MZBs, set this value at 10 pixels to capture outliers, although MZBs will generally not exceed 2 pixels on successive frames that are 5 s apart.</li>
			<li>For the minimum number of frames that a particle has to be present to be tracked, set this value at the number of frames in the movie for MZBs (e.g., 240) for a 20 min movie with 12 frames/min. However, if the cells are fast enough to leave the field of view before the end of imaging, then set the minimum number of frames at less than the duration of the movie. For example, with fast-moving T cells, set the minimum number at the equivalent of 5-10 min of imaging. 
			NOTE: Record a macro of these steps (thresholding and MTrack2-kt) to automate this part of the procedure and save time (see Supplemental Coding File). Using the macro to automatically track a movie in ImageJ takes less than a minute.</li>
		</ol>
		</li>
		<li>Copy the output results into a data spreadsheet. Change the format for decimal display for all spreadsheet cells to zero places after the decimal. Add an empty row at the top of the cell tracks in the spreadsheet and delete the &#8220;D&#8221; column. Save the file as a &#8220;tab stop-separated&#8221; .txt file for subsequent analysis in the Ibidi Chemotaxis tool. 
		NOTE: The MTrack2 plugin outputs the cell tracks in rows of 75 parallel columns. For analysis in the Ibidi chemotaxis tool, the cell tracks must be in a single column. To convert the output, the MTrack2 plugin was modified to output either in the original format or as a single column (Figure 3B) (see Supplemental Coding File &#8220;MTrack2 kt&#8221;, which is written in Java).&#160;Copy the .java file to the ImageJ plugins folder. In ImageJ, select &#8220;Plugins&#160;&#62;&#160;Compile and Run&#34;. Restart ImageJ, and the modified version of MTrack2 should appear in the Plugins menu.</li>
	</ol>
	</li>
	<li>To analyze cell tracks with the Ibidi Chemotaxis tool, download the Ibidi Chemotaxis and Migration Tool v2.0 (&#8220;stand alone&#8221;) and open the program.
	<ol>
		<li>Select &#8220;Import data&#8221; and navigate to the .txt cell track files. Multiple files can be selected at the same time. Imported files appear in the &#8220;Datasets&#8221; pane in red. Select all of them and enter the correct values in the &#8220;Initialisation&#8221; menu below to apply the following settings.</li>
		<li>Enter either the exact number or a range of the number of slices (the number of frames in the movie).</li>
		<li>In the Calibration menu (X/Y calibration), enter the size of a pixel. Locate this information in the properties file of the movie. For MZBs, a 10x objective was used, giving a pixel size of 1.14 &#181;m. In &#8220;Calibration menu | Time interval&#8221;, enter the length of time between frames in the movie.</li>
		<li>Apply the settings. Select &#8220;Plot data&#8221; to view the track plots and confirm that the track files will open. From this point, many options are available which are described in the documentation for the program, including marking the tracks with different colors based on different properties and displaying the individual or average values for velocity, forward migration index, directness, and more (Figure 3B). 
		NOTE: The MZBs take about 10 min to detect and respond to the flow, presumably by polarizing their integrin complexes to the flow-forward point of the cell. A graph of migration index over time will show an initial drop below zero, corresponding to the first couple of minutes that the cell is exposed to flow, followed by a gradual rise upwards until the forward migration index values are positive. Thus, it may be optimal to exclude these first minutes in the final calculations, unless this process is of interest.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

We describe here a method for analyzing the migration of cells that detect the force of shear flow and respond by altering their migration. An analysis of MZBs showed that MZBs migrate spontaneously on ICAM-1 and in the presence of flow, will migrate up the flow. In our previous work, we showed that MZBs do not migrate up the flow on VCAM-1 but instead remain fixed in place. The murine splenic marginal zone contains mainly ICAM-1, while the red pulp contains both ICAM-1 and VCAM-1. From these data, it could be inferred t...

Immune cells are the most motile cells in the human body and often must contend with shear force from blood and lymph flow. However, there are comparatively few studies on shear force-induced migration of leukocytes1,2,3,4,5. We present here a reliable and quantitative protocol to analyze the response of an immune cell to flow in vitro. Performing the assay does not require fabrication of components, and all equipment and consumables are commercially available. The protocol, including cell purification and migration analysis, can be performed in a single day. Finally, although we describe the migration of marginal zone B cells (MZBs), the protocol can be adapted to analyze migration against flow of other types of immune cells. Therefore, it is feasible to use this assay to systematically analyze a broad range of leukocytes with a comprehensive panel of conditions.
MZBs are a population of B cells that, in the mouse, are found only in the spleen and shuttle between the interior of follicles and the marginal zones6,7,8,9. The marginal zone is a layer of immune cells approximately 5&#8211;10 cells thick. The cell layer envelops the follicle and consists primarily of MZBs and macrophages but also invariant natural killer T (iNKT) cells, dendritic cells (DCs), and neutrophils, among others10. The cells in the marginal zone are exposed to unidirectional blood flow originating from splenic arteries that terminate in a marginal sinus surrounding the follicle. The blood flows from holes in the marginal sinus through the marginal zone and is then collected in venous sinuses in the red pulp and restored to the circulation11. The free-flow of blood washes over the MZBs and exposes them to antigens carried in the blood. The MZBs carry the antigen into the follicle by shuttling automatically between the marginal zone and inside of the follicle, which is not exposed to blood. Thus, as MZBs shuttle towards the follicle, they must migrate up the shear force of the blood flow12 (Figure 1A).
In this protocol, we describe how to quantitatively determine how immune cells such as MZBs respond to either no flow or high flow in vitro, in order to reveal how they are programmed to migrate in vivo. In the first step, MZBs are purified from a mouse spleen using magnetic beads coupled to antibodies from commercially available kits. The freshly isolated MZBs are introduced into the well of a flow chamber slide, allowed to settle onto integrin ligands, and exposed to the flow of migration buffer using a pump system (Figure 2A). The cells are imaged using a time-lapse video microscopy system. The images are then processed for analysis with a free ImageJ plugin, MTrack213,14, to automatically track the cells. Tracks can then be quantified with the free Ibidi Chemotaxis tool15 to determine various parameters including velocity, straightness, and migration index. These values can be used to determine the effects of migration inhibitors, cell stimulators, chemokines, and other migration-affecting chemicals on the shear-flow induced migration in order to understand the forces controlling immune cell movement in vivo.

<table><tbody><tr><td>VWR Cell Strainer, 70 &amp;micro;m</td><td>VWR</td><td>10199-656</td><td></td></tr><tr><td>Pre-Separation Filters, 30 &amp;micro;m</td><td>Miltenyi</td><td>130-095-823</td><td></td></tr><tr><td>MZB and FOB cell isolation kit</td><td>Miltenyi</td><td>130-100-366</td><td></td></tr><tr><td>B220 CD45R, clone RA3-6B2, FITC</td><td>Biolegend</td><td>103206</td><td></td></tr><tr><td>CD21 / CD 35, clone 7G6, APC</td><td>BD Biosciences</td><td>558658</td><td></td></tr><tr><td>CD23, clone B3B4, PE</td><td>Biolegend</td><td>101608</td><td></td></tr><tr><td>HBSS</td><td>Biochrom</td><td>L2035</td><td></td></tr><tr><td>D-PBS 1x</td><td>Gibco by Life Technologies</td><td>14190-094</td><td></td></tr><tr><td>BSA albumin fraction V, fatty acid-free</td><td>Roth</td><td>&quot;0052.3&quot;</td><td></td></tr><tr><td>ICAM-1</td><td>R&amp;amp;D Systems</td><td>796-IC-050</td><td></td></tr><tr><td>Ibidi &amp;micro;-slides VI 0.4, hydrophobic, uncoated</td><td>Ibidi</td><td>80601</td><td></td></tr><tr><td>Perfusion set, white, 50 cm, 0.8 mm</td><td>Ibidi</td><td>10963</td><td></td></tr><tr><td>Ibidi Pump system</td><td>Ibidi</td><td>10902</td><td></td></tr></tbody></table>

analysis of shear flow-induced migration of murine marginal zone b cells in vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

We used the protocol outlined above to compare migration of MZBs on ICAM-1-coated slides without flow (0 dyn/cm2) and exposed to shear flow (4 dyn/cm2). Cells were tracked automatically with MTrack2, and the resulting track files were overlaid on the cell migration movies of no flow (0 dyn/cm2) and (4 dyn/cm2) to show the distribution and shape of the tracks (Figure 4A). Cell tracks were then imported into the Ibidi...

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Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

analysis-shear-flow-induced-migration-murine-marginal-zone-b-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.4K Views.  OVGU University of Magdeburg. Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Video: Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Bioengineering

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex vivo transmigration system. Group 2 innate lymphoid cells (ILC2) and CD4+ T helper cells were isolated from spleens and lungs of chicken egg ovalbumin (OVA)-challenged BALB/c mice. We confirmed the expression of CCR4 on both CD4+ T cells and ILC2, comparatively. CCL17 and CCL22 are the known ligands for CCR4; therefore, using this ex vivo transmigration method we examined CCL17- and CCL22-induced movement of CCR4+ lymphocytes. To establish chemokine gradients, CCL17 and CCL22 were placed in the bottom chamber of the transmigration system. Isolated lymphocytes were then added to top chambers and over a 48 h period the lymphocytes actively migrated through 3 &#181;m pores towards the chemokine in the bottom chamber. This is an effective system for determining the chemokinetics of lymphocytes, but, understandably, does not mimic the complexities found in the in vivo organ microenvironments. This is one limitation of the method that can be overcome by the addition of in situ imaging of the organ and lymphocytes under study. In contrast, the advantage of this method is that is can be performed by an entry-level technician at a much more cost-effective rate than live imaging. As therapeutic compounds become available to enhance migration, as in the case of tumor infiltrating cytotoxic immune cells, or to inhibit migration, perhaps in the case of autoimmune diseases where immunopathology is of concern, this method can be used as a screening tool. In general, the method is effective if the chemokine of interest is consistently generating chemokinetics at a statistically higher level than the media control. In such cases, the degree of inhibition/enhancement by a given compound can be determined as well.

In this protocol, lymphocytes are placed in the top chamber of a transmigration system, separated from the bottom chamber by a porous membrane. Chemokine is added to the bottom chamber, which induces active migration along a chemokine gradient. After 48 h, lymphocytes are counted in both chambers to quantitate transmigration.

Herein, we present an efficient method that can be executed with basic laboratory skills and materials to assess lymphocyte chemokinetic movement in an ex ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Flow Cytometric Characterization of Murine B Cell Development

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells ...

Elucidating T-Cell Kinetics: Methods for Real-Time Migration Analysis

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign bodies. T cell migration is a complex process that requires the coordination of many signal inputs from the environment and local immune cells, including chemokines, chemokine receptors, and adhesion molecules. Furthermore, T cell motility is influenced by dynamic surrounding environmental cues, which can alter activation state, transcriptional landscape, adhesion molecule expression, and more. In vivo, the complexity of these seemingly intertwined factors makes it difficult to distinguish individual signals that contribute to T cell migration. This protocol provides a string of methods from T cell isolation to computer-aided analysis to assess T cell migration in real-time&#160;under highly specific environmental conditions.&#160;These conditions&#160;may help&#160;elucidate mechanisms that regulate migration, improving our&#160;understanding of T cell kinetics and providing&#160;strong mechanistic evidence that is difficult&#160;to attain through animal experiments. A deeper understanding of the&#160;molecular interactions that impact cell&#160;migration is important to develop improved therapeutics.

Author Spotlight: Understanding Disease Mechanisms Through Real-Time Analysis of T-Cell Migration

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative analysis.

The adaptive immune response is reliant on a T cell&#39;s ability to migrate through blood, lymph, and tissue&#160;in response to pathogens and foreign ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

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Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

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Study of Cell Migration in Microfabricated Channels

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Assessment of Lymphocyte Migration in an Ex Vivo Transmigration System

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

Flow Cytometric Characterization of Murine B Cell Development

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells