V294.PART II，Chapter 2 Boyden法体外趋化试验

PART II：BASIC CELL MIGRATION AND RELATED ASSAYS

vol.294 Guan J.-L. (ed.) Cell Migration-Developmental Methods and Protocols Chapter 2

Boyden Chamber Assay

Summary

The Boyden chamber assay, originally introduced by Boyden for the analysis ofleukocyte chemotaxis, is based on a chamber of two medium-filled compartments separatedby a microporous membrane. In general, cells are placed in the upper compartmentand are allowed to migrate through the pores of the membrane into the lowercompartment, in which chemotactic agents are present. After an appropriate incubationtime, the membrane between the two compartments is fixed and stained, and the numberof cells that have migrated to the lower side of the membrane is determined. Therefore,the Boyden chamber-based cell migration assay has also been called filtermembrane migration assay, trans-well migration assay, or chemotaxis assay. A numberof different Boyden chamber devices are available commercially. The method describedin this chapter is intended specifically for measuring the migration of Madin-Darbycanine kidney cells using a 48-well chamber from Neuro Probe, Inc.

Key Words: Boyden chamber assay; migration assay; cell motility; cell migration;chemotaxis; hapatotaxis.


1. Introduction

The Boyden chamber, originally introduced by Boyden for the analysis ofleukocyte chemotaxis (1), is ideally suited for the quantitative analysis of differentmigratory responses of cells, including chemotaxis, haptotaxis, andchemokinesis. Random cell motility is generally described as chemokinesis,which is distinguished from directed cell motility toward increasing concentrationsof soluble attractants, such as growth factors (chemotaxis), or along aconcentration gradient of extracellular matrix (ECM) proteins (for haptotaxissee ref. 2). For the induction of chemotactic or haptotactic response of cells,attractants are added to the lower compartment of the chamber. However, for the induction of chemokinesis, equal concentrations of the agent are added onboth sides of the membrane.

In addition, the use of the Boyden chamber-based motility assay has otheradvantages. First, it allows one to have versatility in conducting motility experiments.For example, one can examine the effect of an inhibitor that is specificfor an intracellular signaling molecule or a functional blocking antibody that isspecific to a cell surface protein on cell motility by adding it to the upper chamber,in which cells are loaded. Actually, this type of experiment has been widelyused to examine the potential involvement of a particular intracellular signalingpathway or cell surface protein in cell motility in response to various stimuli(3,4). Second, the Boyden chamber assay is relatively time saving and allowsfor cell-motility analysis on a basis without consideration of the effect from cellproliferation. The time allowed for cells to migrate through a porous membranein the Boyden chamber is generally within a few hours (4–6 h), which is muchshorter than the time required for cells to proceed through a cell cycle (5). Therefore,consideration of the effect of cell proliferation on the results is generallynot necessary. Third, the Boyden chamber assay allows for cell motility analysiswithout consideration of the effect from cell–cell interactions. Cells, in particularepithelial cells, have to release cell–cell contacts for their translocationover a clearly measurable distance. It is already known that cell–cell interactionsand cell migration are controlled largely by different mechanisms (6–9). Because some extracellular factors or genes may be able to stimulate cell migrationbut fail to disrupt cell–cell interactions, it is reasonable to analyze cell–cellinteractions and cell motility separately (10). The Boyden chamber assay is conductedon a basis largely independent of cell–cell interactions. Next, the Boydenchamber assay allows one to measure the relative cell migration rates of cellpopulations transiently transfected with genes of interest. With an appropriatemarker, such as β-galactosidase, which allows the migrated cells to be visualizedby X-gal staining, the relative cell motility of a transiently transfected cellpopulation can be measured (11). Finally, it is easy for learners to gain acquaintancewith the assay and the results are generally very reliable from one experimentto another.

A number of different Boyden chamber devices are available commercially.They basically vary in sample size and quantitation method. In general, cellmigration is quantified by simply enumerating migrated cells under a lightmicroscope or by measuring optical density values of stained cell extracts.Once an appropriate chamber has been chosen, the migration experiments willneed to be optimized for any given cell type and attractants. Some of theimportant factors to be considered are the number of cells to load on the chamber,the pore size of the membrane, the type and concentration of the attractant,and the incubation time. Note that the ECM protein can be coated on the membrane or directly loaded into the lower chamber in its soluble form. In ourexperience, the results derived from these two loading methods are similar. Itis likely that the soluble form of the ECM protein will coat the membrane toform a matrix during the course of the experiment. If a soluble attractant suchas growth factor is used, the ECM protein should still be loaded to allow cellsto attach. Collagen and fibronectin are usually chosen respectively for epithelialcells and fibroblasts to attach. The following protocol is intended specificallyfor measuring the migration of Madin–Darby canine kidney (MDCK)epithelial cells using a 48-well chamber from Neuro Probe, Inc., and solublecollagen as an attractant. However, this could easily be adopted to analysis ofother cell types in response to other stimuli.

2. Materials

1. Standard 48-well chemotaxis chamber (see Fig. 1), including: a lower chamber,a silicone gasket, an upper chamber, and thumb nuts (Neuro Probe; Cabin John,MD).

2. Poretics® polycarbonate: polyvinylpyrrolidone-free, 8-μm pore size, 25 × 80 mm(cat. no. K80SH58050; Osmonics; Livermore, CA).

3. MDCK cells (ATCC; cat. no. CCL-34).

4. Dulbecco’s modified Eagle’s medium (DMEM), high glucose, pH 7.4.

5. Fetal bovine serum (FBS).

6. Versene: 0.2 g of ethylene diamine tetraacetic acid (0.53 mM) and 0.01 g of phenolred in 1 L of phosphate-buffered saline, pH 7.4.

7. 2.5% (w/v) Trypsin (cat. no.15090-046; Gibco Invitrogen): dilute 1:25 in Versenebefore use.

8. Collagen from calf skin (cat. no. C9791; Sigma-Aldrich): dissolve collagen at 1mg/mL in 0.1 N acetic acid. Allow to stir at room temperature until dissolved(takes 1–3 h). Keep collagen stock at 4°C.

9. Methanol.

10. Giemsa stain, modified solution (cat. no. GS500; Sigma-Aldrich): dilute 1:10 indistilled H2O before use.

11. Peri dishes.

12. Glass slide and cover glass (32 × 24 mm).

13. Nail polish.

14. Terg-A-Zyme? (cat. no. 1304; Alconox; New York, NY): an enzyme activedetergent available from Fisher. Dissolve 8 g/L in distilled H₂O.

Fig. 1. Components of Neuro Probe standard 48-well chemotaxis chamber.

3. Methods

3.1. Preparing the Cells

1. Seed 5 × 105 MDCK cells per 60 mm-dish in DMEM supplemented with 10%FBS and penicillin-streptomycin and allow them to grow until 50–70%confluency, which usually takes 18 to 24 h.

2. Remove the medium and wash the cells twice with Versene. Add 1 mL of Versenecontaining 0.05% trypsin and allow the culture to stand at 37°C for 10 to 15 min.

Add 3 mL of DMEM with 10% FBS, pipet the cells off the dish, and transferthem to a 15-mL centrifuge tube.

3. Pellet the cells by centrifugation at 150–200g for 5 min. Remove the medium,add 5 mL of DMEM, and centrifuge again.

4. Remove the medium and resuspend the cells in 1.5 mL of DMEM. Count andadjust the cells to 5 × 105 cells/mL in DMEM (see Notes 1 and 2). If necessary, addthe appropriate concentration of an inhibitor or antibody to the cell suspension.

3.2. Loading and Assembling the Chamber

1. Dilute collagen stock in DMEM to 10 μg/mL before loading (see Note 3). Add30 μL of the diluted collagen or control reagents to each well of the bottom chamber.

The volume should be a slight positive meniscus when the well is filled. Ifnot, re-set the pipetor and load again. It is recommended to use a manual p200pipetor for loading (see Note 4).

2. Use forceps to handle polycarbonate membranes. Cut off 1 mm of the corner of amembrane. Lift the membrane by the end using two forceps, and orient it to thechamber so the cut corner corresponds to the Neuro Probe trademark on the lowerright corner of the chamber (see Note 5). Gently place the membrane (shiny sidefacing up) over the wells of the chamber. Avoid too much movement of the membraneafter it has been placed on the wells.

3. Place the silicone gasket over the membrane with cut corner on the lower right.

4. Place the top chamber over the gasket with the Neuro Probe trademark orientedto the lower right corner. Push the top chamber down against the bottom chamberwith even pressure on all sides with one hand; with the other hand tighten thethumb nuts.

5. Resuspend the cells by gentle mixing and load 50 μL of cell suspension to eachwell of the top chamber. Hold the pipetor vertically so that the end of the pipettip rests against the side of the well just above the membrane. Eject the fluid out of the pipet tip with a rapid motion to avoid trapping bubbles in the bottom ofthe well.

6. Wrap the whole chamber in plastic and incubate it at 37°C and 5% CO2 for 6 h.

3.3. Staining the Membrane

1. Transfer 20 mL of methanol to a Petri dish.

2. Remove the thumb nuts while pressing down the chamber evenly on all sides.

3. Disassemble the chamber, lift the membrane with forceps, and immediately flowit on methanol at room temperature for 10 min to fix cells. The side of the membranewith the migrated cells faces down.

4. Remove the membrane from methanol and allow it to air dry (few min).

5. Dilute 2 mL of Giemsa stain in 20 mL of distilled H2O in a Petri dish.

6. Flow the membrane (the side with migrated cells facing down) on the dilutedGiemsa stain solution for 1 h to stain the cells (see Note 6).

7. Destain the membrane by briefly (few seconds) rinsing it in distilled H2O.

8. Drain the excess H2O from the membrane and place it (the side with migratedcells facing down) on the Petri dish cover. Use a damp cotton swab to wipe theunmigrated cells from the top of the membrane.

9. Cut the membrane into four pieces, each contains 12 wells. Keep track of itsorientation by cutting the lower right corner.

10. Attach the membrane with a little amount of nail polish to a glass slide and coverit with a square cover glass.

3.4. Counting the Cells

1. Survey the stained membrane under a light microscope at 50X and 200X magnificationand select at least three wells for each experimental group on which themigrated cells are well stained and evenly distributed.

2. Take micrographs using a digital camera (Nikon COOLPIX995) connected tothe microscope at 50X magnification. Each micrograph covers a well on the membranesee Note 7).

3. Remove the camera from the microscope and connect to a computer using theUSB cable. Acquire the images from the memory card of the digital camera andview the images with the aid of the software Adobe Photoshop? (Adobe Systems;San Jose, CA). Select the function “view/show” to add grids on the imageand count the number of cells on the monitor (see Fig. 2). Alternative, adjust thesize of the image for printing on an A4 size paper and then count the number ofcells on the printed image.

4. Calculate the value (mean ± standard error) of the migrated cells from at leastthree wells for each experimental group.

Fig. 2. A representative micrograph of a stained membrane taken by a digital cameraunder a light microscope.

3.5. Cleaning the Chamber

1. After disassembling the chamber, immediately rinse all parts in running tap H2Ofor 30 min. Rinse the chamber components several times with distilled H2O andlet them air dry at room temperature.

2. For periodic cleaning, soak all the chamber components in Terg-A-Zyme ^® solutionat 55°C for 1 h. Wash them thoroughly under running tap H2O for 30 minand rinse them several times with distilled H2O. Soak them in distilled H2O overnightand allow them to air dry (see Note 8).

4. Notes

1. It is recommended that less than six experimental groups are planned for a chamber,which allows at least eight wells for an experimental group. This ensures atleast three representative wells that can be selected for quantification for eachexperimental group. In addition, more experimental groups will take more timefor preparation, which causes cells in different experimental groups to stay insuspension with varied time, which may affect cell motility.

2. Because the volume varies from one pipetor to another, it is better to prepare alittle more sample for loading. For example, if eight wells per experimental groupare planned for loading, prepare enough sample for loading 10 wells. The volumeof cell suspension (5 × 105 cells/mL) to load on a well of the upper chamberis 50 μL, that is, 2.5 × 104 cells per well. Make 2.5 × 105 cells in 0.5 mL ofDMEM for loading 10 wells.

3. Collagen is less soluble in neutral pH than in low pH. After diluting collagen inDMEM, immediately load it to the lower chamber.

4. To avoid trapping bubbles in the wells of the chamber, the liquid should not beexpelled completely from the pipet tip. In addition, for the lower chamber, it is important to load the correct volume of attractants, which should form a slightpositive meniscus when the well is filled. For loading the upper chamber, holdthe pipetor vertically so that the end of the pipet tip is against the side of the welljust above the membrane and expel the liquid quickly from the pipet tip.

5. Ensure to keep track of the orientation of the membrane by cutting it on the rightlower corner.

6. Instead of immersing the membrane in Giemsa stain, we let the membrane flow onthe stain solution. This allows only the side of the membrane with the migratedcells to be stained.

7. With the aid of a digital camera and computer, the total number of stained cellsin a well can be counted accurately and objectively. Directly count the cells inselected fields from a well under a microscope at high magnification is not recommended.

8. It is important to immediately immerse all parts of the chamber in water afterdissembling the chamber and wash them thoroughly to prevent the accumulationof debris. It is recommended that after approximately five experiments, the chambercomponents are cleaned with Terg-A-Zyme? (see Subheading 3.5.2.). Neverautoclave the chamber or immerse it in water hotter than 60°C.

Acknowledgments

The author would like to thank Po-Chao Chan and Chun-Chi Liang for technicalassistance. Work in the author’s laboratory is supported by the NationalScience Council, Taiwan.

References

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