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 defo....

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 ....

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 apply f.......

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

List of materials used in this article

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