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

Summary

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.

Abstract

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.

Introduction

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 leukocytes^1,2,3,4,5. 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 ....

Protocol

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

1. Isolate leukocytes.
   1. Sacrifice an 8 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 µm cell strainer mes....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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