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, 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.
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 5–10 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.
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
2. Flow Experiment
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.
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 Chemotaxis tool (ICT) to generate track plots of each movie (Figure 4B). The average migration index (called "FMIy" in the ICT), velocity, straightness (called "directness" in the ICT), and straight-line distance (called "Euclidean distance" in the ICT) of cell tracks from 4 movies each for both conditions were calculated in the Chemotaxis tool using the "measured values" command. These average values were then copied into GraphPad Prism for generating graphs and calculating statistical significance (Figure 4C).
Figure 1: MZB shuttling. (A) Model of MZB shuttling between the marginal zone and the follicle in the spleen. MZBs require internalization of S1PR1, a receptor for S1P, and functional CXCR5, a receptor for the CXCL13 chemokine, in order to enter the follicle. Additionally, to reach the follicle, MZBs must migrate against the force of blood flow emanating from pores in the marginal sinus that envelops the follicle. If a MZB loses adhesion to ICAM-1, the ligand for LFA-1 integrin, it is pushed into the red pulp by the force of the flow, where increased amounts of VCAM-1, the ligand for VLA-4, would not support migration. (B) Flow cytometry gating strategy to test purity of sorted MZBs using antibodies against B220, CD23, and CD21. Please click here to view a larger version of this figure.
Figure 2: Setup of the fluidics system, pump, microscope, and incubation chamber. (A) Image of the pump system (Ibidi18) consisting of a pump with connected fluidics unit and laptop running the software to control the pump. Higher magnification image: the flow chamber slide with 6 flow chambers, the first of which is attached to the tubing of the fluidics unit. (B) Typical setup for measuring in vitro migration of MZBs against flow. A microscope equipped with a heating chamber (black box) containing the fluidics unit connected to a pump (blue square object to the lower right of the microscope) outside the microscope. Higher magnification image: the fluidics unit inside the microscope heating chamber. Please click here to view a larger version of this figure.
Figure 3: Quantification of imaging data. (A) Representative images of a frame from a movie of migrating MZBs before (left) and after (right) thresholding the image to convert the cells to black objects on a white background. Scale bars in both low and high magnification images = 100 µm. (B) Left panel: image of typical results output from the MTrack2 (single column output) plugin. Right panel: image of the Ibidi Chemotaxis and Migration 2.0 tool after input of MTrack2 results showing a track plot of migrating MZBs and a "measured values" display window showing the averages of parameters including velocity, migration index, and directness (green boxes), among others. Calibration settings shown include the pixel resolution of 1.14 µm per pixel and a time interval of 5 s (0.083 min) per movie frame. Please click here to view a larger version of this figure.
Figure 4: Example of results using the protocol to measure MZB shuttling. (A) Representative stills of migrating MZBs with an overlay of tracks from the ImageJ plugin "MTrack2 kt". Note: the track colors were arbitrarily set by the "Manual Tracking" plugin for ImageJ. Scale bars in both low and high magnification insets = 100 µm. (B) Representative track plots of migrating MZBs from the Ibidi Chemotaxis and Migration 2.0 tool. Red lines and black lines represent cell tracks that terminate below or above the horizontal axis, respectively. In (A) and (B): left, MZBs migrating with no flow (0 dyn/cm2); right, MZBs migrating to a 4 dyn/cm2 flow. (C) Migration index (FMIy), velocity, straightness (directness), and distance for MZBs migrating with no flow (0 dyn/cm2) or flow (4 dyn/cm2) (n = 4 mice in 4 separate experiments). Error bars = mean ± SD; Student's t test, **p < 0.01, ***p < 0.001. Please click here to view a larger version of this figure.
Supplemental File 1: MTrack2_kt.java. Please click here to download this file.
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 that MZBs would migrate up the flow while in the marginal zone but not in the red pulp. The analysis was validated in vivo using MZBs with defective adhesion that were mislocalized to the splenic red pulp by the force of the flow12. For these reasons, MZBs represent a good positive control for testing the flow migration system described here, even if the system is used to study a different cell type.
The most critical aspect of the procedure is ensuring consistency in cell handling on the slide. Because the quantifiable aspects of cell migration depend on the degree to which the cells adhere to the slide, any decrease of cellular adhesion would make reproducible results difficult. Decreased cellular adhesion could result from inconsistency in multiple factors, including temperatures of the migration buffer or the heated incubation box (cold reduces adhesion), levels of cations in the cell suspension (cations affect integrin binding), slide handling (tapping or flexing the slide could dislodge cells), and cell density (collisions between cells could affect migration parameters). Other sensitive points during the procedure include not pipetting the buffer into the slide wells with too much force prior to attaching the tubing (as this could reduce cell adhesion) and keeping the time required for protocol steps consistent in every experiment (cell settling time could affect adhesion). In summary, any possible influence on cell adhesion must be kept consistent throughout repetitions to produce reliable cell migration data.
This method is easy to set up since it uses commercially available equipment and supplies and none of the steps require advanced instruction. For analysis of MZB, as well as other cell types such as cultured CD8+ T cells, coating the slides with various integrin ligands is sufficient to observe the migration up the flow. Studying flow-induced migration response to various integrin ligands leads to direct identification of active integrins on the cell surface, possibly revealing mechanisms relevant to in vivo functions as with MZB migration up the flow on ICAM-1 but not VCAM-1. However, it is also possible to add an endothelial cell layer to the flow chambers. One example of immune cell migration that is affected by shear flow is T cell extravasation through endothelial layers3. This procedure was used to unravel the activation of T cell adhesion via integrins, selectins, and chemokines, and to model lymphocyte migration through the blood-brain barrier. The only limitation to the cells that can be analyzed with this method is that they must be able to sense the force of flow as a directional signal.
Although the method outlined here is used to characterize cellular behavior such as velocity and turning, it can also be extended to analysis of the molecular aspects of migration. Molecular complexes relevant to flow-induced migration, including integrins such as LFA-1 and their cytoskeletal adaptors, would be located within 200 nM of the surface of the slide, amenable to visualization with TIRF microscopy. This addition to the method would be ideal for studying cells from mice with mutations in integrin- or cytoskeleton-related proteins. As many immune cells migrate at various stages in their development through blood or lymph flows, the in vitro migration described can be used to systematically test many kinds of cultured or primary leukocytes for response to flow and reveal how the cells are programmed to migrate in an immune response in vivo. In conclusion, the assay described here provides a reliable and straightforward method for analyzing flow-induced migration of MZBs that can also be extended to other cell types.
The authors have nothing to disclose.
This work was supported by grants from the "Deutsche Forschungsgemeinschaft" SFB 854/TP11 to K.-D.F.
VWR Cell Strainer, 70 µm | VWR | 10199-656 |
Pre-Separation Filters, 30 µm | Miltenyi | 130-095-823 |
MZB and FOB cell isolation kit | Miltenyi | 130-100-366 |
B220 CD45R, clone RA3-6B2, FITC | Biolegend | 103206 |
CD21 / CD 35, clone 7G6, APC | BD Biosciences | 558658 |
CD23, clone B3B4, PE | Biolegend | 101608 |
HBSS | Biochrom | L2035 |
D-PBS 1x | Gibco by Life Technologies | 14190-094 |
BSA albumin fraction V, fatty acid-free | Roth | "0052.3" |
ICAM-1 | R&D Systems | 796-IC-050 |
Ibidi µ-slides VI 0.4, hydrophobic, uncoated | Ibidi | 80601 |
Perfusion set, white, 50 cm, 0.8 mm | Ibidi | 10963 |
Ibidi Pump system | Ibidi | 10902 |