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>

<ol>
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P., Theodoly, O., Gucciardi, A., Hogg, N., Lellouch, A. 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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 &#181;m</td>
			<td>VWR</td>
			<td>10199-656</td>
		</tr>
		<tr>
			<td>Pre-Separation Filters, 30 &#181;m</td>
			<td>Miltenyi</td>
			<td>130-095-823</td>
		</tr>
		<tr>
			<td>MZB and FOB cell isolation kit</td>
			<td>Miltenyi</td>
			<td>130-100-366</td>
		</tr>
		<tr>
			<td>B220 CD45R, clone RA3-6B2, FITC</td>
			<td>Biolegend</td>
			<td>103206</td>
		</tr>
		<tr>
			<td>CD21 / CD 35, clone 7G6, APC</td>
			<td>BD Biosciences</td>
			<td>558658</td>
		</tr>
		<tr>
			<td>CD23, clone B3B4, PE</td>
			<td>Biolegend</td>
			<td>101608</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Biochrom</td>
			<td>L2035</td>
		</tr>
		<tr>
			<td>D-PBS 1x</td>
			<td>Gibco by Life Technologies</td>
			<td>14190-094</td>
		</tr>
		<tr>
			<td>BSA albumin fraction V, fatty acid-free</td>
			<td>Roth</td>
			<td>&#34;0052.3&#34;</td>
		</tr>
		<tr>
			<td>ICAM-1</td>
			<td>R&#38;D Systems</td>
			<td>796-IC-050</td>
		</tr>
		<tr>
			<td>Ibidi &#181;-slides VI 0.4, hydrophobic, uncoated</td>
			<td>Ibidi</td>
			<td>80601</td>
		</tr>
		<tr>
			<td>Perfusion set, white, 50 cm, 0.8 mm</td>
			<td>Ibidi</td>
			<td>10963</td>
		</tr>
		<tr>
			<td>Ibidi Pump system</td>
			<td>Ibidi</td>
			<td>10902</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...

Watch this Scientific Journal Video about Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro at JoVE.com

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

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

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

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 ...

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

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 ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...