A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

Summary

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 µm from the gel surface.

Abstract

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 µm in diameter, in PA gels, within 1.6 μm of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Introduction

The mechanical interaction of a living cell with its local environment has commonly been studied using PA gels. These substrates rely on a simple, well-characterized protocol established by Dembo and Wang in 1997¹. One of the main advantages of these substrates is that their stiffness may be tuned by modifying the concentrations of specific components of the gel solution. This provides a desirable platform to study cells’ interaction with environments of different rigidities. When PA gels are coated with extracellular matrix (ECM) proteins, cells adhere to them, generating force. As a result of cell force, the gel deforms as an elastic body. This deformation depends on the magnitude of the force applied by the cells and the elastic properties of the gel. Various studies have employed PA gels to investigate cellular traction forces.

In one variation of PA gel fabrication, fluorescent microspheres (beads) are embedded throughout the gel to quantify cell traction forces on gels of different rigidities². Upon cell force application, the beads displace from their initial location following gel deformation. The deformation field is measured from the individual bead displacements. This deformation field is utilized with elasticity theory and the elastic properties of the gel to compute the traction forces. These measurements provide insight as to how cells mechanically sense and interact with their local microenvironment³.

In many widely used PA gel fabrication protocols, the beads are intermixed throughout the PA gel in its liquid, unpolymerized state. A fully polymerized PA gel contains fluorescent beads throughout its volume. When computing cell traction forces, beads most near the gel surface (cell-substrate interface) are monitored. The displacements of these beads are assumed to occur on the cell culture surface for simplicity in the force calculation. The actual location of the beads within the depth of the gel is disregarded. However, in an elastic medium (such as PA gel), a bead closer to a point of force application will move more than a bead that is further away from the point. Thus, treating the displacement of a point (at the bead location) distal from the surface as that at the surface results in an underestimation of cellular tractions. The degree of error depends on the distance of the bead from the surface. The error cannot be estimated without the knowledge of the location of the bead.

The need for a simple method to confine beads very near the cell culture surface has been addressed by a few techniques. One way is to increase the density of beads throughout the whole gel such that there are a sufficient number of beads in the top focal plane to measure motion very near the surface. Another technique involves building a confocal imaging chamber for live cell imaging such that the light from only the beads in the upper-most focal plane is collected⁴. A different method involves overlaying an extremely thin layer of PA gel containing beads on top of an already polymerized gel without beads⁵. A drawback of each of these techniques is that the precise location of the beads within the gel is not known. This introduces error into the calculation of the displacement field of the beads, and thus the calculation of cell forces. Another technique involves conjugation of beads to the top surface of an already polymerized PA gel using Sulfo-SANPAH⁶. This technique ensures the beads are indeed only on the top of the PA gel, but the extent to which they are embedded in the depth of the gel is unknown. This could potentially create a local topography for the cells, which could alter cell behavior, as prior work has suggested that cells can sense force several microns away⁷. Recently, a technique for patterning PA gels with 1 μm diameter fluorescent fibronectin dot markers in a regularized array was established⁸. In this case, the depth of the fluorescent markers is known, and is essentially zero, as the fibronectin pattern is indirectly printed on the gel surface. However, this method does not provide a continuous environment on which cells can attach, as the ECM protein is limited to 1 μm diameter dots. A method for fully integrating tracker beads within PA gels and confining them to a known location very near the surface has yet to be established.

Here, we develop a technique to constrain sub-μm to μm diameter fluorescent beads to a focal plane very near the cell culture surface within PA gel. A gel is typically cured by sandwiching unpolymerized liquid gel solution between two glass plates. One of the plates is functionalized so that the gel strongly adheres to it. The other is unfunctionalized and is removed after the gel cures. We modify this removable glass surface by coating it with a layer of beads. Upon sandwiching the liquid gel between the functionalized and the bead coated glass surface, the beads transfer to the gel while it is curing. This limits the distance of the beads’ integration into the gel within 1.6 µm of the surface. Glass-bottom Petri dishes are used as the adherent surface on which the gel is cured. To form a flat top gel surface during polymerization, a circular glass cover slip is used to sandwich the gel with the glass-bottom Petri dish. Prior to gel fabrication, the top glass cover slip is coated with poly-D-lysine (PDL), yielding a positive surface charge. The PDL is blown off with compressed air, and a solution of beads in water is deposited on the cover slips. We utilize carboxylated fluorescent microbeads, which carry a negative charge, and interact with the positively charged surface created by treatment with PDL. After blowing the bead solution off the coverslip with compressed air, a single layer of beads remains electrostatically coupled to the dry cover slip. The PDL coating does not affect the adhesiveness of the glass to the gel surface, as the glass slides are undamaged and removed from the PA gel fully intact.

The glass-bottom Petri dishes are made adherent by treatment with 97% 3-aminopropyl-trimethoxysliane and 0.5% glutaraldehyde. PA gels of desired rigidities are created by mixing appropriate concentrations of bisacrylamide and acrylamide via a standard procedure⁹. A droplet of the gel solution is pipetted onto the glass-bottom Petri dish. The glass cover slip containing the beads is used to sandwich the gel with the Petri dish. When the gel is cured, the top cover slip is removed leaving the beads embedded in the PA gel within 1.6 µm from the surface.

Protocol

Fabricating and functionalizing PA gels of varying stiffnesses with fluorescent microspheres embedded near the cell culture surface.

1. Functionalizing the Top Glass Cover Slips

1. Clean glass cover slips (#1.0, 12 mm diam.) with soap and water, followed by ethanol to remove extraneous dust.
2. Place glass cover slips on a grated surface (i.e. pipette tip holder) such that they are not touching to facilitate ease of interaction with the coverslips.
3. Coat the entire surface of the cover slips with Poly-D-Lysine (0.1 mg/ml) for 1 hr (Figure 1A).
4. During this time, perform a 1:10,000 dilution of the colloid solution of 0.1 μm diameter, red fluorescent microspheres with deionized (DI) water to obtain a particle density of approximately 1 microsphere per 20 μm² on the gel surface. See Figure 2 for the results of various dilutions. This dilution can be modified to meet the need of specific experiments.
5. Place the diluted solution in an ultrasonic water bath for 30 min.
6. After 1 hr, use tweezers to carefully lift each cover slip and blow dry with air. Return the dry cover slips to the grated surface.
7. Remove the diluted colloid solution from the ultrasonic bath and pipette 150 μl onto each cover slip. Leave for 10 min (Figure 1B).
8. Use tweezers to carefully lift each cover slip and blow dry with air. Return the dry cover slips to the grated surface and store in the dark until ready to use.

2. Preparing PA Gel Directly on Glass Bottom Petri Dishes

1. Preheat hotplate to 100 °C.
2. Lay out the desired number of glass bottom Petri dishes (35 mm dish with 14 mm micro-well, #1.0) on a flat surface in a chemical fume hood.
3. Cover the glass portion of each Petri dish micro-well with 97% 3-aminopropyl-trimethoxysliane (3-APTES) for 7 min for chemical activation. Take caution to avoid inadvertent dripping of the 3-APTES to the surface of the plastic in the Petri dish to avoid degradation of the polystyrene.
4. After 7 min, fill the Petri dish with DI water and dispose into waste container.
5. Repeat step 2.4 3x for each dish, and then shake the Petri dish to remove extra water. Place the Petri dishes on the hot plate until the glass portion is dry.
6. Remove the Petri dishes from the hot plate and return to a flat surface in a chemical fume hood.
7. In a chemical fume hood, make a solution of 0.5% glutaraldehyde and cover the glass portion of each Petri dish well with the solution for 30 min. Take caution to avoid inadvertent dripping of the glutaraldehyde to the surface of the plastic in the Petri dish to avoid degradation of the plastic.
8. After 30 min, fill the Petri dish with DI water and dispose into waste container to rinse and remove the glutaraldehyde.
9. Repeat step 2.4 3x for each dish, and then shake the Petri dish to remove extra water. Place the Petri dishes on the hot plate until the glass portion is dry.
10. Before mixing the components of the PA gel solution, move the functionalized glass slides into the chemical fume hood such that they are easily accessible, allowing for the quick sandwiching of the gel with the glass bottom Petri dishes after mixing the gel solution.
11. In a 15 ml centrifuge tube, mix 40% bisacrylamide, 2% acrylamide, and acrylic acid in immediate succession in the concentrations listed in Table 1 (adapted from published protocol¹⁰) to achieve the desired matrix elasticity.
12. Add 100 mM HEPES, 10% ammonium persulfate, and TEMED in quantities listed in Table 1 corresponding to desired matrix elasticity to complete the gel solution.
13. Immediately pipette 15 μl of gel solution onto the center of the glass portion of the petri dishes.
14. Immediately pick up a functionalized glass cover slip with tweezers.
    1. Flip the glass cover slip over such that the fluorescent beads are on the side making contact with gel solution.
    2. Lay the cover slip gently on top of the now-liquid PA gel such that the functionalized side is in contact with the gel (Figure 1C). Note: For best results, a second person is recommended for the role of adding the cover slip in order to avoid possibility of partial polymerization while pipetting liquid PA gel solution onto multiple Petri dishes.
15. Flip all Petri dishes over to assist with avoiding gravity effects on fluorescent nanoparticles polymerizing into lower levels of the PA gel.
16. Wait for at least 35 min, or until the stock solution of PA gel has visibly polymerized in its centrifuge tube.
17. Flip the Petri dishes back over and fill them with PBS to assist with removing the cover slip.
18. Carefully make contact with the glass portion of the Petri dish and the outline of the cover slip, using tweezers to scrape the circumference of the cover slip. Perform several cycles until the cover slip is dislodged. Remove the cover slip and dispose in a proper sharps waste container.
19. After removing all cover slips, fluorescent beads will have transferred to the gel (Figure 1D). Cover the PA gels completely with PBS, place the Petri dish lid on each dish, and store at 4 °C.

3. Functionalizing PA gel with Fibronectin

1. Prepare the following premixed solutions as described in an established protocol¹¹: Soak solution (137 mM NaCl, 5% (v/v) glycerol) and 2x conjugation buffer (0.2 M 2-(N-morpholino)ethanesulfonic acid (MES), 10% (v/v) glycerol, pH 4.5).
2. Use a vacuum pump in a biological hood to remove all PBS from the glass-bottom dishes containing the PA gels.
3. Pipette soak solution onto each gel such that the gel is completely submerged. Incubate at room temperature for at least 1 hr.
4. Warm 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) to room temperature.
5. Mix 10x solutions of EDC (150 mM, 19 mg/ml in DI water) and NHS (250 mM, 29 mg/ml in DI water).
6. Mix 1 part 10x EDC, 1 part 10x NHS, 3 parts DI water, and 5 parts 2x conjugation buffer.
7. Use a vacuum pump in a biological hood to remove the soak solution. Make sure all fluid is removed from the gel surface.
8. Add enough NHS/EDC solution to cover the gel surface and fill the glass-bottom well of the Petri dish (150–250 μl). Incubate at room temperature for 30 min in the dark.
9. Thaw fibronectin at room temperature. Once thawed, mix sterile DI water to create a 50 μg/ml fibronectin solution.
10. Use a vacuum pump in a biological hood to remove the NHS/EDC solution. Make sure all fluid is removed from the gel surface.
11. Add 150 μl of fibronectin solution to each gel. Incubate at room temperature for 35 min to allow for attachment of fibronectin.
12. After 35 min, add PBS to each Petri dish and store at 4 °C for up to 2 weeks.

4. Traction Force Experiments

1. Warm cell media, PBS, and trypsin to 37 °C in a water bath.
2. Rinse gels 5x with sterile PBS, aspirating the PBS in between rinses, and leave covered in the hood.
3. Add 1 ml trypsin per 25 cm² to flask containing cells. After cells are lifted from flask, dilute the trypsin with cell media and count the cells. Based on gel surface area, determine the number of cells required per gel for a final cell seeding density of 3,000 cells/cm². Dilute or concentrate suspension such that 150 μl of the cell-media mixture contains this number of cells. Aliquot 150 μl of cell suspension on to each gel.
4. Place the Petri dishes containing cells in an incubator for 30 min. Then, carefully remove the Petri dishes and fill the remainder of the Petri dish with media (approximately 2 ml) such that the surface of the dish is completely submerged.
5. Place the Petri dishes back in the incubator until image acquisition.
6. Prepare the microscope and data acquisition system for imaging: insert the 40x water immersion objective, insert the Differential Interference Contrast (DIC) prism, select the mCherry or equivalent fluorescent filter, and turn on the environmental chamber.
7. When prepared for imaging, remove one Petri dish from the incubator and place it gently on the microscope stage. Remove the Petri dish lid for DIC imaging.
8. Locate a single cell and capture a single still image of the cell in DIC.
9. Without moving the microscope stage, switch the imaging mode to fluorescence. Focus on the fluorescent microspheres and record an image of the microspheres.
10. Carefully remove cell media from the Petri dish with a pipette and add 0.05% trypsin-EDTA.
11. Image the microspheres under the cell after the cells have detached.
12. Use particle image velocimetry (PIV) analysis in ImageJ¹² to compute the displacement field due to cellular forces.

Results

Confocal imaging was used to determine that the beads were indeed underneath the gel surface and to quantify their precise location within the gel depth. Fluorescent beads of a different wavelength than those inside the gel were allowed to settle on the surface, and the distance between the embedded fluorescent nanoparticles and those on the surface was calculated using a centroid identification algorithm. The location of the bead on the gel surface serves as a reference for the top surface of the gel—where cells a...

Discussion

When using this technique, it is essential that the bead solution is properly diluted and the beads are chosen based on desired diameter, PA gel substrate stiffness, and the size scale of the phenomena that is explored in the desired experiment.

Caution should be taken when diluting the bead solution prior to functionalizing top glass cover slips. The spacing between beads on the gel surface can be altered by changing the dilution factor of the colloid solution. Diluting the solution too much ...

Materials

Name	Company	Catalog Number	Comments
Reagents
97% 3-Aminopropyl-trimethoxysliane (APTES)	Sigma-Aldrich	281778
70% Glutaraldehyde	Polysciences, Inc.	111-30-8
1 M HEPES buffer solution	Sigma-Aldrich	83264
40% Acrylamide	Sigma-Aldrich	A4058
2% Bisacrylamide	Sigma-Aldrich	M1533
0.1 µm Fluorescent microspheres (mCherry)	Invitrogen	F8801
Fibronectin, Human, 1 mg	BD Biosciences	354008
Ammonium persulfate	Bio-RAD	161-0700
Tetramethylethylenediamine (TEMED)	Bio-RAD	161-0801
Poly-D-lysine	Millipore	A-003-E
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)	Thermo Scientific	22980
N-hydroxysulfosuccinimide (NHS)	Thermo Scientific	24500
0.05% Trypsin-EDTA (1X)	Life Technologies	25300-054
NaCl	Sigma-Aldrich	S9888
Acrylic acid	Sigma-Aldrich	147230
Glycerol	Sigma-Aldrich	G7757
2-(N-morpholino)ethanesulfonic acid (MES)	Sigma-Aldrich	M3671
Materials
35 mm Glass bottom dish with 14 mm micro-well #1 cover glass	In Vitro Scientific	D35-14-1-N
Glass cover slips, 12 mm diam.	Ted Pella, Inc.	26023