Name: analysis of shear flow-induced migration of murine marginal zone b cells in vitro
Uploaded: 2018-11-26T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro at JoVE.com

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%...

<ol>
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P., Theodoly, O., Gucciardi, A., Hogg, N., Lellouch, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+lymphocytes+orient+against+the+direction+of+fluid+flow+during+LFA-1-mediated+migration.">T lymphocytes orient against the direction of fluid flow during LFA-1-mediated migration.</a> Biophysical Journal. 104 (2), 322-331 (2013).</li><li>Woolf, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymph+node+chemokines+promote+sustained+T+lymphocyte+motility+without+triggering+stable+integrin+adhesiveness+in+the+absence+of+shear+forces.">Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces.</a> Nature Immunology. 8 (10), 1076-1085 (2007).</li><li>Cinamon, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine+1-phosphate+receptor+1+promotes+B+cell+localization+in+the+splenic+marginal+zone.">Sphingosine 1-phosphate receptor 1 promotes B cell localization in the splenic marginal zone.</a> Nature Immunology. 5 (7), 713-720 (2004).</li><li>Cinamon, G., Zachariah, M. A., Lam, O. M., Foss, F. W., Cyster, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicular+shuttling+of+marginal+zone+B+cells+facilitates+antigen+transport.">Follicular shuttling of marginal zone B cells facilitates antigen transport.</a> Nature Immunology. 9 (1), 54-62 (2008).</li><li>Cyster, J. G., Schwab, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine-1-phosphate+and+lymphocyte+egress+from+lymphoid+organs.">Sphingosine-1-phosphate and lymphocyte egress from lymphoid organs.</a> Annual Reviews in Immunology. 30, 69-94 (2012).</li><li>Schwab, S. R., Cyster, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+a+way+out:+lymphocyte+egress+from+lymphoid+organs.">Finding a way out: lymphocyte egress from lymphoid organs.</a> Nature Immunology. 8 (12), 1295-1301 (2007).</li><li>Cerutti, A., Cols, M., Puga, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marginal+zone+B+cells:+virtues+of+innate-like+antibody-producing+lymphocytes.">Marginal zone B cells: virtues of innate-like antibody-producing lymphocytes.</a> Nature Reviews Immunology. 13 (2), 118-132 (2013).</li><li>Mebius, R. E., Kraal, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+spleen.">Structure and function of the spleen.</a> Nature Reviews Immunology. 5 (8), 606-616 (2005).</li><li>Tedford, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+opposing+forces+of+shear+flow+and+sphingosine-1-phosphate+control+marginal+zone+B+cell+shuttling.">The opposing forces of shear flow and sphingosine-1-phosphate control marginal zone B cell shuttling.</a> Nature Communications. 8 (1), 2261 (2017).</li><li>ImageJ. MTrack2 Available from: <a target="_blank" href="https://imagej.net/MTrack2">https://imagej.net/MTrack2</a> (2018)</li><li>. 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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, ...

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