V294.PART II，Chapter 5 细胞粘附实验（英文版）

PART II：BASIC CELL MIGRATION AND RELATED ASSAYS

vol.294 Guan J.-L. (ed.) Cell Migration-Developmental Methods and Protocols Chapter 5

Cell-Adhesion Assays

Dennis F. Kucik and Chuanyue Wu

Summary

One of the most important properties of cells that are derived from multicellularorganisms is their ability to adhere to extracellular matrix proteins or other cells. Analysisof cell–extracellular matrix and/or cell–cell adhesion, therefore, is of importantvalue to experimental biologists as well as clinical investigators. Over the past severaldecades, many different cell-adhesion assays have been developed. Based on theexperimental conditions, most of the cell-adhesion assays fall into two categories,namely static adhesion assays and flow adhesion assays. Static assays are widely usedto assess the adhesion of many types of cells (e.g., epithelial cells and fibroblasts) tothe extracellular matrix. The flow adhesion assays are more appropriate for analysis ofblood cell (e.g., leukocyte) adhesion to endothelial cells, to each other, or extracellularmatrix proteins. This chapter describes two basic protocols, one for analysis of celladhesion under static conditions and the other for measurement of cell adhesion undershear stress. In addition, variations to the basic protocols and areas where special attentionis required for successful application of these methods are discussed.

Key Words: Cell adhesion; integrins; extracellular matrix; fibronectin; N-acetyl-β-D-glucosaminidase; enzyme-linked immunosorbent assay; flow; shear stress.

1. Introduction

Cell adhesion is a fundamental process that is critically involved in embryonicdevelopment and diseases. On the basis of the experimental conditionsunder which adhesion is measured, methods for measurement of cell adhesioncan be in general divided into two types. In the first type of the methods, celladhesion is analyzed under static conditions. Static assays are widely usedto assess the adhesion of different types of cells, including epithelial cellsand fibroblasts. They are relatively simple to perform and provide a valuableassessment of the adhesiveness of cells to a defined extracellular matrix substrate (e.g., fibronectin). However, static assay methods poorly simulate adhesionthat occurs in blood, or even lymph vessels, under shear stress. Therefore,a second method is provided to measure cell adhesion under shear stress, whichcan be achieved by using flow chambers. The use of a flow chamber enablesthe researcher to simulate blood flow to reconstruct cell systems in the presenceof shear. Some adhesive events occur only under shear and, thus, cannotbe characterized under static conditions (1,2). Flow chambers are widely availablecommercially and allow the researcher to introduce cells between two flatsurfaces under conditions of laminar flow while the process of adhesion isvisualized using a microscope (3). These assays are most commonly used tostudy leukocyte adhesion, either with endothelial cells or to substrates of purifiedligands. They are also useful to study bacterial adhesion (4). An additionaladvantage of using flow chambers for adhesion assays is that they producewell-defined forces, in contrast to some of the wash methods of static assays.

Flow cell assays can also detect rapid events, so that adhesion events, such asremodeling of contacts during rolling, can be studied on a time scale as smallas a fraction of a second (5,6). Furthermore, the entire process of adhesioncan be observed, as a leukocyte progresses from rolling to firm adhesion andfinally migration through the endothelium (7). In this chapter, we first describea basic protocol of static adhesion assay. Next, we provide a protocol for measuringcell adhesion under shear stress. Finally, we discuss variations to thebasic protocols and areas where special attentions are required for successfulapplication of the methods.

2. Materials

2.1. Static Adhesion Assays

1. 96-Well enzyme-linked immunosorbent assay (ELISA) plates (Corning).

2. Extracellular matrix proteins (fibronectin, laminin, collagens, or other extracellularmatrix proteins, either alone or in combination).

3. PBS: 2.7 mM KCl, 137 mM NaCl, 1 mM KH2PO4, 10 mM Na2HPO4, pH 7.4.

4. Bovine serum albumin (BSA; 10 mg/mL in PBS; denatured at 85°C for 10 minprior to use).

5. α Minimum essential medium (α-MEM; Life Technologies).

6. Substrate solution: 3.75 mM p-nitropheno-N-acetyl-β-D-glucosaminide (Sigma)in 50 mM citrate buffer, pH 5.0 (aliquoted and stored at –20°C).

7. Stop solution: 5 mM ethylene diamine tetraacetic acid (EDTA), 50 mM glycinebuffer, pH 10.4.

2.2. Measurement of Adhesion in Shear Stress

1. Cultured endothelial cells or substrate coating materials (fibronectin, gelatin, orother extracellular matrix proteins, either alone or in combination).

2. Adhesion media (primary = Hanks Balanced Salt Solution [HBSS], alternate =serum-free culture media with 10 mM HEPES buffer).

3. BSA.

4. In vitro flow chamber.

5. Inverted phase-contrast microscope.

6. Low power (approx 10–20X) objective.

7. Programmable syringe pump.

8. 25-mL Syringes.

9. Video camera.

10. VCR.

11. TV monitor.

12. Video cables.Recommended:

13. Fluorescence optics.

14. Stage incubator. Optional:

15. Computerized image digitization system.

16. Motion analysis software.

3. Methods

3.1. Static Adhesion Assay

The following protocol described has been successfully used for analyses ofChinese ovary hamster cell adhesion to fibronectin (8). With minor modifications,it can be used to analyze static adhesion of almost any cell types.

1. Coat wells of 96-well ELISA plates with 10 μg/mL fibronectin in PBS at 37°Cfor at least 1 h (see Note 1).

2. Incubate each well with 200 μL of heat-denatured 10 mg/mL BSA in PBS at37°C for at least 1 h.

3. Rinse the wells twice with α-MEM.

4. Harvest Chinese hamster ovary cells with 0.3 mM EDTA in PBS, rinse the cellsthree times with α-MEM, and suspend them to a final density of 3 × 105 cells/mL(see Note 2).

5. Add 100 μL of the cell suspension to each well of the fibronectin-coated ELISAplates and incubate for 60 min in a 37°C incubator under a 5% CO2–95% airatmosphere (see Note 3).

6. Rinse the wells three times with PBS (see Note 4).

7. Add 60 μL of the substrate solution to each well (see Note 5).

8. In parallel experiments, add 1 mL of the cell suspension to a 1.5-mL microfugetube that is precoated with heat denatured 10 mg/mL BSA in PBS. Pellet the cellsimmediately by centrifugation in a microfuge at 3800g for 15 min and then keepthe cell pellets in –20°C freezer. Simultaneous to step 7, add 600 μL of the substratesolution to the microtube, vortex, and then transfer the cell/substrate mixtureto new wells of the 96-well ELISA plates (60 μL/well; see Note 6).

9. Incubate the plates at 37°C in 100% humidity until color develops.

10. Add 90 μL of the stop solution to each well of the 96-well ELISA plates.

11. Measure absorbance at 405 nm (A405 nm) using a microplate reader.

12. Determine cell adhesion using the formula: cell adhesion (%) = A405 nm of theadhered cells (steps 1–7) divided by A405 nm of the “total” cells (step 8) ×100%(see Note 6).

3.2. Measurement of Adhesion in Shear Stress

The subsequent methods described outline 1) preparation of adhesive substrates,2) loading of cells with fluorescent marker, 3) the flow assay, and 4)data analysis.

3.2.1. Preparation of Adhesive Substrates

Commercial flow chambers incorporate either the surface of a culture dishor a cover slip as one of the two parallel plates between which laminar flowoccurs. This surface should be coated with either endothelial cells or extracellularmatrix proteins as an adhesive substrate. This is described in three alternateprocedures (cell monolayer preparation, steps 1–4; purified substrateon small part of dishes, steps 5–8; and purified substrate on cover slip, steps9–18).

3.2.1.1. CELL MONOLAYER PREPARATION

1. Incubate plastic tissue culture dishes or glass cover slips with 1% gelatin in PBSfor 30 min at room temperature.

2. Remove gelatin solution.

3. Add endothelial cells to result in a cell density of approx 5000 cells/cm2. Transferto 37°C incubator for culture.

4. Feed cells every 2–3 d until confluent. Depending on the cell type, cells are usuallysuitable for assays for 2–3 d after they reach confluency (see Note 7).

3.2.1.2. PREPARATION OF PURIFIED SUBSTRATE ON PLASTIC DISHES

Purified ligands can be used instead of endothelial cell monolayers for awell-defined adhesive substrate. Because leukocytes and endothelial cells useseveral sets of adhesion molecules simultaneously to establish adhesion, purifiedligand substrates may be necessary to isolate a particular adhesion receptor-ligand interaction. Purified substrates also allow the researcher to varyproperties such as ligand density, which is difficult to do with whole cells. Thefollowing procedure is designed to coat only a small portion of the plastic dishto conserve reagents.

1. Outline an area to coat with a marker or diamond stylus. If a diamond stylus isused, keep the scratches shallow so as not to interfere with sealing of flow chambergaskets.

2. Add 25 μL of coating solution (10 μg/mL matrix protein, such as fibronectin orcollagen, in PBS, pH 7.4) and incubate at room temperature for 1 h.

3. Remove coating solution and add 1 mL of 1% w/v BSA in PBS. Incubate at roomtemperature for 1 h.

4. Remove BSA solution. Wash twice with PBS.

3.2.1.3. PREPARATION OF PURIFIED SUBSTRATE ON GLASS COVER SLIPSCovalently linking matrix molecules to adsorbed poly-L-lysine (PLL), as inthe following protocol, results in tighter, more uniform binding to cover slipsthan direct adsorption. If, however, you prefer to avoid PLL, direct adsorptionof matrix materials, as in the plastic dish protocol, may provide an adequatesubstrate. Cover slips precoated with PLL are also commercially available.

1. Sterilize the cover slips by autoclaving, ultraviolet light, or flaming with ethanol.

2. Place cover slips in 35-mm tissue culture dishes or multi-well plates.

3. Outline an area to coat with a marker or diamond stylus. If a diamond stylus isused, keep the scratches shallow so as not to interfere with sealing of flow chambergaskets.

4. Add enough PLL (100 μg/mL in PBS, pH 7.4) to coat the outlined area. We usePLL MW 70K-150K (Sigma Chemical; St. Louis, MO). Incubate at room temperaturefor 10 min.

5. Wash cover slips three times with PBS.

6. Add 1% glutaraldehyde and incubate at room temperature for 30 min.

7. Wash cover slips three times with PBS.

8. Add 25–100 μL of coating solution (depending on the size of the cover slip) at 10μg/mL and incubate at room temperature for 1 h.

9. Add 1% BSA (enough to completely cover the cover slip) to block. Incubate atroom temperature for 30 min.

10. Wash cover slips once with PBS. Add fresh PBS to keep moist until used forflow assay.

3.2.2. Loading Cells With Fluorescent Dye

Although rolling and adherent cells can be visualized without fluorescence,the increase in contrast afforded by labeling the perfused cells greatly facilitatesobservation and counting, and is sometimes a requirement for object trackingsoftware.

The following protocol uses BCECF-AM as a fluorescent marker. We haveused this dye extensively and find it to have no adverse effects on adhesion.Several other cell markers will also work, but concentrations and incubationtimes may have to be adjusted.

1. Wash cells in HBSS, then resuspend at 1–2 × 106/mL in 25 mL of HBSS.

2. Add BCECF-AM (Molecular Probes; Eugene, OR) to make 1 μM (from 2 mMstock in DMSO).

3. Incubate for 30 min at room temperature in the dark.

4. Wash the cells three times with 35 mL of HBSS.

5. Resuspend in HBSS to a concentration of 0.5 × 106 cells/mL.

3.2.3. Flow Assay

In the past, investigators constructed their own laminar flow chambers, butthis is no longer worthwhile unless special characteristics are required. Severalflow chambers are now available commercially, and, in general, they allwork well. The same basic methods are used to conduct flow cell adhesionexperiments regardless of the particular flow chamber used. The primary differenceamong apparatuses is how the substrate (either a cell monolayer orpurified ligand) is incorporated. The following method usually assumes use ofa Glycotech flow chamber (Glycotech; Rockville, MD), in which cells aregrown in a tissue culture dish that becomes part of the flow chamber uponassembly. However, this protocol can be easily adapted for use with otherchambers. Simply modify the assembly procedure according to manufacturer’sinstructions, and use cell- or ligand-coated glass coverslips instead of coateddishes if required.

1. Turn on the stage incubator and warm adhesion buffer prior to experiment (ifexperiments are to be done at 37°C; see Note 8).

2. Load the cells to be perfused with a fluorescent dye (optional; see Subheading3.2.2. dye-loading protocol).

3. Load leukocytes or other cells to be perfused into a syringe, if using the “pushing” method, or a tube or other reservoir, if using the “pulling” method (seeNote 9).

4. Attach the tubing to the syringe and remove any air bubbles from the system.

Assemble the flow chamber, according to manufacturer’s instructions, incorporatingan adhesive substrate of either cells or purified ligand. (Cells or ligand willbe pre-coated on either a tissue culture dish or a cover slip, depending on themanufacturer of the flow chamber.) Work out any bubbles in the system that mayhave formed during the assembly process.

5. Program the syringe pump to give the desired flow according to manufacturer’sinstructions (see Note 10).

6. Mount the flow chamber on the microscope, focus the system, and adjust theoptics, camera, and any recording equipment (see Note 11).

7. Choose an area to observe (see Note 12), start the recording equipment, andbegin the flow. Record 3–6 min of data.

8. Stop the flow (to conserve cells) and move to a new area of observation to collectmore data. In general, several areas of observation are required to collect enoughdata for statistical significance. It is a good idea to resuspend the cells at thispoint. If using the “pushing” method, with the cells in the syringe, rotate thesyringe 180°. If using the “pulling” method, drawing the cells from a tube orother reservoir, resuspend by gently shaking or swirling the tube or reservoir.

3.2.4. Data Analysis

3.2.4.1. QUANTIFYING ADHERENT CELLS

The raw data from flow cell adhesion experiments will be movies of cells asthey interact with an adhesive substrate, either other cells or purified matrix materials.

These interactions can be quantified in a number of ways. It is important tokeep in mind that the method of analysis should be tailored to the scientific questionsto be answered. For example, if you are interested only in the number ofadherent cells, a simple count of adherent cells/min can be obtained by visualreview of videotapes. Even when using simple, low-tech methods of analysis,though, it is important to have precise definitions of the quantities measured. Forexample, under shear stress, cells are rarely completely stopped. Therefore, a reasonabledefinition of firmly adherent cells might be, “cells that moved less than1 cell diameter in 5 s.” There is some variability in the literature as to the actualnumbers used to define firm adhesion, and this often reflects differences inexperimental conditions (higher shear stresses and less adhesive substrates leadto briefer adhesion). To enable comparison of studies, however, a quantitativecriterion should be chosen and clearly specified in materials and methods.

The number of adherent cells per minute will depend on the size of the fieldobserved, so this number must be normalized for area of observation. Area canbe calculated using a stage micrometer, which is a glass slide with accurate distancescales etched into the surface. These can be purchased from several scientificsupply companies (e.g., Fisher Scientific; Pittsburgh, PA). Simply imagethis known length standard on your system, and use it to calibrate distances (inmicrons) on the monitor.

The following protocol can be performed with nothing other than a VCR andmonitor. It can be made far less tedious, however, if the sequences are digitizedinto AVIs with a video capture card. Many inexpensive cards are available forthis purpose. This will give the investigator greater control over functions suchas rewind and freeze frame. Some loss of both temporal and spatial resolutionusually occurs in this process, however, so be sure that you choose a card thatproduces movies of sufficient quality to reliably distinguish adhesion events.

1. Choose a video segment of defined length (e.g., 1 min) to analyze.

2. Visually review the segment, counting the number of cells that stop and markingtheir locations with a lab marker. If the number is large, it may help to use amarker to divide the screen into sections and count each separately.

3. Review the segment again to be sure that each cell that seemed to stop meets yourquantitative criterion for firm adhesion.

3.2.4.2. QUANTIFYING ROLLING CELLS

More care must be taken if rolling cells are to be identified visually. This isbecause of the properties of laminar flow, fluid in a flow chamber moves more slowly near the chamber walls. Because the adhesive substrate constitutes onewall of the chamber, cells carried by hydrodynamic flow near the substrate,without any adhesive interactions, may appear to be rolling (13). An objectivemeasure developed to identify rolling cells is the critical velocity (14). This isbased on the fact that adhesive interactions will substantially decrease cellvelocity, distinguishing it from a cell that is moving in the slowest hydrodynamicflow. Knowing the size of the cell, the viscosity of the fluid (near that ofwater for simple buffers), and the shear stress, an upper limit of velocity compatiblewith rolling can be calculated. Therefore, even if your experimentalquestion deals only with the number of rolling cells, and not their velocity, it isa good idea to calculate a critical velocity for a few cells to verify that cellscounted as rolling do indeed have adhesive interactions with the substrate. Onceyou know what to look for, it may then be feasible to identify rolling cellsvisually.

A simple calculation for critical velocity is:

V_critical = β · r · γ

where r = the radius of the cell, γ = the shear rate, and β is a drag factor. Areasonable estimate of β that can be used for cells in a flow chamber is 0.5.

Cells moving slower than the critical velocity can be confirmed as being rollingcells.

3.2.4.3. ROLLING VELOCITIES

A simple calculation of rolling velocity can be performed by marking aknown length (in microns) on the monitor (again, calibrated using a stagemicrometer) and measuring the time required for a cell to move this distance.

The temporal resolution of this method will be limited, since times less thana few seconds may be difficult to measure. Greater time resolution can beachieved using professional-quality videotape machines that can accuratelycount frames, or by digitizing the sequences. Alternatively, more accuratevelocity determinations can be made using more sophisticated image processingsoftware that can accurately localize objects in each image and convertpixels to distances. A variety of commercial software packages areavailable to track cell motion, and most of these will work for tracking rollingcells. NIH Image, available online for free, will also track object motion(Website: http://rsb.info.nih.gov/nih-image/about.html).

4. Notes

1. Different extracellular matrix proteins (laminin, collagen I) or their fragmentscan be used to coat 96-well plates. Although 10 μg/mL is often a good startingpoint, the concentrations of the coating solutions should be optimized for differentmatrix proteins and cells under study.

2. If cells are not effectively detached by treatment with EDTA alone, solutionscontaining both EDTA and trypsin (e.g., solution containing 0.05% trypsin and0.53 mM EDTA from Life Technologies) could be used. In this case, cells shouldbe washed at least once with serum-containing medium after harvesting with thetrypsin-EDTA solution, followed by washing with serum-free medium (see step4). The number of cells added to each well should be adjusted for different celltypes and it should not exceed the maximal number of cells that can adhere to thecoated extracellular matrix protein in a well. The maximal number of adheredcells can be estimated by adding a different number of the cells to each well. After incubating the cells in a 3°C incubator under a 5% CO2–95% air atmospherefor a prolonged period of time (e.g., 2 h), the wells are examined under amicroscopy and the number of cells that form monolayer reaching 100%confluence represents the maximal number of cells that can adhere to the coatedextracellular matrix protein in a well.

3. Depending on the purpose of the experiments, cells can be treated with variousagents (e.g., inhibitory or activating anti-integrin antibodies) before the additionof the cells to the wells. The length of incubation should also be optimized basedon the purpose of the experiments, the cell type and the extracellular matrix protein. Samples should be analyzed in duplicate or triplicate under each experimentalcondition.

4. The simplest way to remove unattached cells is by washing the wells with a bufferas described in the basic protocol. Even, gentle force should be applied duringthe wash to avoid washing away adhered cells. Alternatively, unattached cellscan be removed by centrifugation (9).

5. In the basic protocol, the number of cells is quantified by measuring the activity ofN-acetyl-β-D-glucosaminidase, a ubiquitous lysosomal enzyme, using p-nitropheno-N-acetyl-β-D-glucosaminide as a substrate. This method is sensitive and producesa linear relationship between A405 nm and the cell number for many differenttypes of cells over a wide range of cell numbers (10). Alternatively, the number ofcells can be quantified by staining the cells with dyes such as Crystal Violet (11).

6. Alternatively, the numbers of the total cells and adhered cells in a well can beestimated by photographing multiple (>3) randomly selected microscopic fieldsbefore and after the washes and counting the cell number. Cell adhesion (%) canbe presented as: the number of the adhered cells (after wash) divided by the numberof the “total” cells (before wash).

7. The quality of the monolayer will depend on the cell type and the initial coatingdensity. While a monolayer can be established more quickly by coating at higherdensity, coating at a lower density and allowing more time may result in a moreorderly monolayer. After the cells are confluent, they may change properties suchas shape and adhesion molecule expression. It is a good idea to always plate cellsat the same density and do the experiments on the same day post-confluency. Optimal conditions for each cell type should be worked out empirically. Thechoice of plating cells on plastic vs glass will largely depend on the flow chamberselected. Some flow chambers incorporate glass cover slips as one wall of the chamber, while others use a plastic tissue culture dish. Growing cells in tissue cultureplastic generally results in superior adhesion as compared to glass cover slips, evenif both are coated with extracellular matrix materials. The advantage of glass coverslips, however, is superior optical properties. Birefringence of plastic can be a problemif Nomarski imaging is used. Plastic also tends to have background fluorescence.

Analysis of adhesion under flow does not generally require optimal opticalconditions, though, so visualization through plastic is usually adequate for thispurpose.

Endothelial cells generally need to be stimulated to express the adhesion moleculesnecessary for leukocyte adhesion. Whether, and how, you choose to stimulateyour endothelial cells, however, will depend on the physiologic conditionsyou are mimicking. Interleukin-1, tumor necrosis factor-α, and lipopolysaccharidehave been considered good paradigms for proinflammatory mediators andare often used experimentally on cultured endothelial cells to simulate inflammation(12).

8. Many investigators perform experiments at room temperature, although adhesionmay be stronger at 37°C. The choice of temperature will depend, amongother things, on the experimental question, and how important metabolic processesare to the aspect of adhesion being studied.

9. The optimal cell concentration depends on the experiment. Cells should be denseenough that sufficient events are observed in the few minutes of recording. Thiswill depend on the adhesiveness of the cells being studied. If the cell concentrationis too high, however, cell behavior could be dominated by collisions withother rolling or adherent cells. We find that 0.5 × 10⁶ cells/mL works well formany types of leukocytes. Remember, for meaningful comparison of experiments,the number of cells perfused per minute must be the same under all experimentalconditions. This requires that cells be loaded into the syringe or reservoir at aconsistent density and not be allowed to settle out of suspension. A syringe pump can be used either to push or pull cells through a flow chamber. To push, the syringe itself can be filled with cells. However, many investigatorsprefer to attach the syringe to the outlet side of the chamber to pull fluid and cellsthrough the system. An advantage of the pulling method is that the cells can be keptin a tube and warmed in a water bath. Also, the tube can be shaken or swirledperiodically to keep the cells in uniform suspension. Both warming and mixing canbe much more problematic if the cells are in a syringe mounted on a syringe pump.
For most experiments, a syringe size of 10–25 mL works well. Larger syringesenable longer periods of uninterrupted flow, but smaller syringes can producesmoother flow, especially at low flow rates. The number of cells required to do anexperiment will depend on the rate of flow and the length of observation needed togather sufficient data to answer the particular experimental question.

10. The desired flow will depend on the experiment to be performed, but generallyshould result in a physiologic shear stress. Simulation of arterial flow will requirehigher shear rates than simulation of venous or post-capillary venule conditions. Typically, physiologic shear stresses are roughly 0.5–5.0 dynes/cm².

Only shear rates and shear stresses can be compared for flow chambers orblood vessels of different geometry; flow rates are meaningless for comparison,because it is the combination of the flow rate and geometry that determine theforces experienced by the cells. Flow chamber manufacturers often provide achart or table to make this conversion. If necessary, shear stress can be calculatedfrom the dimensions of the flow chamber and the rate of fluid flow (13). Werecommend doing a range of shears to determine the shear dependence of anyphenomena observed.

11. Most investigators currently record on a VHS videotape for later digitization.The technology to record directly to computer hard drive at high frame rates nowexists, though, and this may become the standard soon. For some cameras, thismay be the only option. DVD recorders are also available now at reasonable cost,and represent another option. Whatever the recording medium, we recommendmaking a sample recording of a minute or so before starting the experiment tomake sure that all of the cables are connected correctly and that the equipment isworking properly.

12. When using endothelial cells as an adhesive substrate, it is important to assessthe integrity of the monolayer. This can be performed by simply visually examiningthe endothelial cells using bright-field or phase contrast optics. Rips or otherdefects in the monolayer will result in interaction of flowing cells with not onlyendothelial cells, but also with bare plastic or glass, which may make the datauninterpretable.

Acknowledgments

This work was supported by NIH grants GM65188 and DK54639 (to C. Wu)and by a VA Merit Award and NIH grant R21 AI 54552-01 (to D. F. Kucik).

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