The authors would like to acknowledge Drs. Jonathan Reichner and Angle Byrd for cell experiment insight. The National Science Foundation Graduate Research Fellowship Program (GRFP) supported this work.

1. 3D Mold Design and Parts
<ol>
	<li>Mold
	<ol>
		<li>Before working, obtain a silicone elastomer kit, a live cell imaging chamber, a 22 mm glass coverslip, and a machined aluminum metal cube with dimensions 10.07 mm x 3.95 mm x 5.99 mm. Prepare the live cell imaging chamber for molding by placing the coverslip in the bottom holder and assembling the rest of the chamber as directed by the vendor.</li>
		<li>Next, using forceps, place the machined aluminum metal cube in the middle of the live cell imaging chamber housing and on top of the coverslip, and then set aside.</li>
		<li>Mix silicone elastomer solutions according to manufacturer&#8217;s protocol to make 5 ml of elastomer.</li>
		<li>Using a disposable lab spatula, pour silicone elastomer solution into live cell imaging chamber setup and ensure not to move placed machined aluminum metal cube. Place the system on the lab bench, in a safe location O/N for curing.</li>
		<li>The next morning, deconstruct the live cell imaging chamber, as recommended by the manufacturer and pull out mold using forceps. Using forceps, carefully extract the machined aluminum metal cube from mold. Go to the sink and rinse the mold with deionized water. Place the mold on a paper towel to dry.</li>
		<li>Once dry, using a hobby utility knife, cut slits through the mold, spaced 2.34 mm apart from each longitudinal end, inside the silicone mold. Ensure the mold stays in a safe and dry place until you are ready to construct the system for an experiment.</li>
	</ol>
	</li>
	<li>Hydrophilic and Hydrophobic Glass Coverslips Preparation
	<ol>
		<li>To allow the collagen matrix to adhere to a surface, create hydrophilic coverslips:
		<ol>
			<li>Using a disposable pipette, measure out 150 &#181;l of 3-aminopropyl-trimethoxysilane and pour solution in a 50 ml tube. Add 30 ml of 100% ethanol to the 50 ml tube with a second disposable pipette and close the lid. Vortex the solution for 2 min, ensuring complete mixing. Pour solution in a glass Petri dish and set aside.</li>
			<li>Pour 15 ml of 100% ethyl alcohol in a second Petri dish and set aside (make sure to label dishes).</li>
			<li>Using a new disposable pipette, measure out 30 ml of deionized water and pour solution into a 50 ml tube. Next, using a new disposable pipette measure out 1,875 &#181;l of glutaraldehyde and pour solution into the same 50 ml tube and close the lid. Vortex the solution for 2 min, ensuring complete mixing. Pour mixture into third Petri dish, and set aside.</li>
			<li>Using forceps, take out one 22 mm round glass coverslip and rinse both sides with 100% ethanol mixture using a disposable pipette.</li>
			<li>Place rinsed glass coverslip into 3-aminopropyl-trimethoxysilane with 30 ml of 100% ethanol solution dish and allow to sit in solution for 5 min.</li>
			<li>With forceps, take out glass coverslip and rinse again with 100% ethanol solution with disposable pipette.</li>
			<li>Drop rinsed glass coverslip into 1,875 &#181;l of glutaraldehyde and 30 ml of deionized water mixture and set aside for 30 min.</li>
			<li>After 30 min, remove glass coverslip with forceps and rinse with deionized water and place onto dry tissue O/N to dry at RT.</li>
			<li>Repeat coverslip dipping and moving for as many coverslips as needed with the same generated solutions.</li>
		</ol>
		</li>
		<li>Make hydrophobic coverslips by the given protocol.
		<ol>
			<li>Add 500 &#181;l of tridecafluoro-1,1,2,2-tetrahydrooctyl, 100 &#181;l of acetic acid and 19.4 ml of hexane in a 50 ml tube and vortex for 2 min. Pour solution in glass Petri dish and set aside.</li>
			<li>Using clean forceps, take out one glass coverslip and drop into prepared mixed solution for 2 min. After 2 min have passed, take out glass coverslip with forceps and rinse with deionized water using a disposable pipette and place coverslip onto dry tissue O/N to dry at RT. Store coverslip in plastic Petri dishes until used.</li>
			<li>Repeat coverslip dipping and moving for as many coverslips as needed with the same generated solutions.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Mold Assembly
<ol>
	<li>Before using molds, using a disposable pipette rinse the mold with 90% ethyl alcohol over a chemical waste container. Next, place the mold in a Petri dish filled with deionized water and allow to sit O/N.</li>
	<li>Take mold out of solution using forceps and place on a towel to air dry before starting the mold assembly.</li>
	<li>While the mold is air-drying, cut the square hydrophilic coverslips into two rectangles slightly larger than 3.95 mm x 5.99 mm using a high-precision diamond-scribing tool. Slide each cut rectangle coverslip into the slots cut into the silicone mold.</li>
	<li>Flip the mold upside down and apply vacuum grease along the bottom of the silicone mold using a disposable pipette. Next, flip the mold back right-side up and press the bottom of the mold onto the circular hydrophilic glass coverslip to create a seal.</li>
	<li>With disposable gloves, pick up the assembled mold and place into a disposable Petri dish and then into a bio-hood. Turn on UV Bio-hood lamp for 1 hr to sterilize the mold before experimentation occurs.</li>
</ol>
3. Collagen Mixture and 3D Matrix
<ol>
	<li>Before preparing the collagen mixture, stain and prepare cells for imaging according to standard protocols28.</li>
	<li>Using standard laboratory techniques and protocols29,30, in a bio-hood, mix cell media and chemoattractant in a 15 ml tube to desired chemoattractant concentration. Pipette 5 ml of specific cell media-chemoattractant concentration solution into a 15 ml tube and move the solution into a heated water bath.</li>
	<li>Using a pipette, extract 5 ml of just cell media into a second 15 ml tube and place in a heated water bath to heat up the solution for microscopy experiments.</li>
	<li>In a bio-hood, pipette 30 &#181;l of 10x phosphate buffered solution (PBS) into micro centrifuge tube. Add 6 &#181;l of 1 N sodium hydroxide (NaOH) into same micro centrifuge tube and vortex for 15 sec.</li>
	<li>Obtain collagen I rat tail solution from the fridge and move to the bio-hood using standard laboratory techniques and protocols29. Ensure the collagen is still cold. Pipette 168.3 &#181;l of collagen into same micro centrifuge tube.</li>
	<li>Next, add 18 &#181;l of yellow-green fluorescent carboxylate-modified microspheres using a pipette to the same micro centrifuge tube. Vortex the micro centrifuge tube with all the solutions for 30 sec.</li>
	<li>Add 77.7 &#181;l of desired cell media-cell mixture (approximately 2 x 106 cells/ml concentration) into micro centrifuge tube and mix using pipette tip for 20 sec. As necessary, alter the matrix density by following the custom collagen mixture adapted for the addition of fluorescent beads and cell viability table (Table 1).</li>
	<li>Pipette 300 &#181;l of collagen-cell mixture into the center well (well #2) of the prepared mold assembly. Place the mold onto a disposable Petri dish and move it into a standard incubator for 20 min at 37 &#176;C with 5% CO2.</li>
	<li>Remove system from incubator and place back into bio-hood. Using forceps, remove both hydrophobic cover slips by clamping onto the top of each coverslip and pulling up and away from the mold.</li>
	<li>Move the whole system to the desired microscopy and image to ensure collagen mold sides are still straight to allow for a planar diffusion gradient.</li>
	<li>With the system still under the microscope, using a 100 &#181;l single-channel pipette, add 100 &#181;l of cell media into well #1. Next, using a 100 &#181;l single-channel pipette add 100 &#181;l of cell media with desired chemoattractant concentration into well #3.</li>
	<li>Immediately after adding solutions into both wells, commence imaging.</li>
</ol>
4. Imaging and Diffusion Modeling
<ol>
	<li>If one wants to find the diffusion coefficient for the specific collagen density to calculate the specific concentration, follow the protocol below:
	<ol>
		<li>Follow the given protocol above titled &#8220;Collagen Mixture and 3D Matrix,&#8221; instead of generating cell media with desired chemoattractant concentration, make 5 &#181;M rhodamine in a 15 ml tube using standard calculations and protocols30.</li>
		<li>Image the 3D collagen matrices with a confocal system mounted on an inverted microscope using an argon laser (488 nm) to capture the fluorescence. Image every 2 or 3 sec for up to 7 hr.</li>
	</ol>
	</li>
	<li>For Diffusion Modeling: Using the mathematical model shown below, write a computational software code for processing and analysis with these general steps. 
	<img alt="Equation 1" src="/files/ftp_upload/52948/52948eq1.jpg" />
	<ol>
		<li>Import images into computational software for rescaling and normalizing by the maximum intensity. Convert images to a gray color-map of the image to select the edge. Pick an axis along the center of the image, to the right of the center and to the left of the center.</li>
		<li>Using the computational software code, plot the normalized intensity, along with the true intensity and the difference between the two.</li>
		<li>Next, input the parameters necessary for fitting (concentration, time, and length).</li>
		<li>Plot the normalized concentration versus time and the concentration profile across the x-axis with a polynomial fit to the data.</li>
		<li>Calculate the coefficient of diffusion using the computational software code.</li>
	</ol>
	</li>
</ol>
5. Experimental Measurements
<ol>
	<li>Referring back to the diffusion equation, rewrite the equation to find the specific concentration at any location within the system as shown in below. 
	<img alt="Equation 2" src="/files/ftp_upload/52948/52948eq2.jpg" /> 
	Where: 
	L = Length of the collagen matrix 
	x = location under investigation 
	D = coefficient of diffusion 
	t = elapsed time</li>
	<li>During chemotaxis experiments, ensure to record the specific time that the moment chemokine was added to system. Once cell migration movement is found, ensure to record confocal time-lapse recording, and note the specific time and location from edge where imaging occurred.</li>
	<li>During data analysis, plug the noted elapsed time, location and coefficient of diffusion for the specific cell into the equation to find the normalized specific concentration at any point within the collagen for both diffusion and cell experiments.</li>
</ol>
6. Tracking Cell Migration Utilizing TFM
<ol>
	<li>To track cell migration using TFM/DVC techniques follow the protocol below:
	<ol>
		<li>After the addition of desired chemoattractant concentration into well #3, start imaging the 3D collagen matrices with a confocal system mounted on an inverted microscope to find a cell to investigate.</li>
		<li>Once a moving cell is found, place the cell in the middle of the field of view and utilize a piezoelectric motor (to image in the z-axis), image every 1 or 2 min.</li>
		<li>For calculating cell force generation and deformation, generate a computational code to find the displacements of the fluorescence beads following the TFM/DVC protocols31.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

The most critical steps for successful diffusion experiments with or without cells are: correctly setting up the mold assembly; developing the necessary manual dexterity to prevent damage during extraction of hydrophobic coverslips; ensuring to find a very good linear starting line to correctly calculate the diffusion coefficient; correct experimental calculations of both collagen and chemoattractant; correctly use of live cell imaging system to ensure matrix does not dry out; and maintaining a sterile, healthy culture.
When conducting diffusion experiments, it is imperative to make sure the diffusion plots match the traditional shapes of a linear diffusion model (Figure 4).
Running the analysis at several different x-locations on each sample, it is possible to calculate the average coefficients of diffusion for rhodamine in the various concentrations of collagens, as reported in Table 2. These results are consistent with previous research&#8212;coefficient of diffusion through the collagen gel decreases as the concentration of collagen decreased due to an increase of porosity in the matrix.
According to the diffusion model equation at steady state, one expects to have a linear concentration profile across the x values. By fitting a linear function to the points, one can see that they are all, in fact, linear. The R2 value of fitting a linear trend line for each of the best fit lines for the three collagen concentrations were at or above 0.92. The norms of the residuals in all extrapolated plots were .0227, indicating a good fit. This linear relationship is in agreement with the analytical solution. The concentration does not begin at 100%, as would be expected, because the imaging does not begin directly at the edge of the collagen sample. It is also impossible to perfectly calibrate the imaging.
Additionally, we were able to reference other papers that looked at diffusion in gels to compare results. Shenoy and Rosenblatt found the diffusion coefficient of BSA (radius = 4 nm) in a 30 mg/ml succinylated collagen solution to be 2.2 x 10-7 cm2/sec32. The radius of rhodamine is 0.78 nm, which explains its faster diffusion time through collagen24. In diffusion studies, it is common to utilize the radius to compare molecules. As the radius increases, the molecular weight also increases. Ramanujan et al. found the diffusion coefficient of dextran (radius = 6 nm) in polymerized 20% collagen gels (equivalent of 2.0 mg/ml concentration gels) to be 4.72 x 10-8 cm2/sec24. Again, this is comparable to the diffusion coefficient we observed for the 2.0 mg/ml concentration gel (D = 1.28 x 10-6 cm2/sec), if we take into account the molecule size difference. Leddy and Guilak studied the diffusion of dextrans through articular cartilage33. It is possible to compare diffusion through collagen matrices and through articular cartilage because approximately two-thirds of the mass of articular cartilage is polymeric collagen (predominately type II). Leddy and Guilak found the diffusion coefficient of 3 kDa dextran through articular cartilage to be between 6-10 x 10-7 cm2/sec33. Knowing that the system works properly we were then able to calculate the specific concentration anywhere within the system, using sawtooth and transient system heat transfer models. This additional metric can be used during cell experiments to track cell-concentration understanding and gradient slope.
Time duration of experiments may be a limiting factor when using our approach. Therefore, it is important to use a cover for the live cell imaging system to ensure the matrix does not dry out. Using a cover during experiments, we were able to successfully monitor the diffusion of rhodamine up to 7 hr and 4 hr during cell experiments without the gel drying up. If one does not use a cover, the gel might dry up faster and significantly affect the diffusion and migration of cells.
The protocol described herein uses a well-controlled planar diffusion model/system to be able to measure cell forces in a 3D environment during chemotaxis. Here we present a newly developed simple system that can be utilized for TFM/DVC experiments. Using this system, the researchers will be able to: 1) successfully model diffusion through 3D collagen matrices and verify, by fitting the data to analytical expression, that the planar gradient diffusion system, when seeded with cells, should promote chemotaxis and not chemokinesis; 2) calculate the coefficient of diffusion for collagen of five different concentrations; and 3) successfully find the specific concentration of the chemoattractant within the diffusion chamber. With this approaches, one can further examine cell chemotaxis with specific metrics not easily and readily available without the laborious approaches of multifaceted systems and complex diffusion gradients.

The preferred movement of cells towards a concentration gradient, known as chemotaxis, plays an important role in pathological and physiological processes in the body. Such examples are skin and mucosa wound healing1, morphogenesis2, inflammation3, and tumor growth4,5. It has also been shown that cancer cells can migrate through both individual and collective cell-migration strategies6. Moreover, diffusional instability mechanisms can induce the separation of single or clustered cells from a tumorous body/object and then can immigrate towards a source of nutrients and thus invade wider areas and tissues7.
Furthermore, it has been shown that diverse migration mechanisms can be active in 2D and in 3D, due to different roles of adhesion molecules8. Therefore, a move to physiologically relevant in vitro assays to investigate cell motility in a measureable and simple way is of significance in understanding cell movement phenomena9. Unfortunately, the difficulty in analyzing cell migration, a comprehensive quantifiable chemotaxis assay usually requires a long laborious method, founded on the measurement of impartial cell motility and transport phenomena models.
Past experimental approaches to investigate cell chemotaxis include the Boyden chamber10 and the under agarose assay11. However, within these early assays, cell migration experiments did not monitor the movement in respect to time. More, importantly, the concentration gradients used for the experiments were not well defined or completely understood while only sustaining the signaling for no more than a few hr. Furthermore, early chemotaxis chamber attempts restricted cell migration to two dimensions and did not allow one to monitor the kinetics of migration12. Looking at the Boyden chamber, an endpoint assay would not allow the researcher to observe migration visually and could not directly differentiate chemotaxis (directional movement) from chemokinesis (random movement). Additionally, several variables&#8212;differences in the pore size and thickness of membranes&#8212;made the chamber very difficult to easily reproduce and concealed the migrant reaction of cells to chemokines13,14.
With the new understanding of microfluidics, new chambers and micro-devices have been investigated as an instrument to investigate cell locomotion under interstitial flow conditions or chemotaxis15,16. Under these new devices, new cell metrics were introduced and investigated, like the effect of shear stress on a cell17,18. Unfortunately, past and current microfluidic chemotaxis chambers limited studies of cell migration to 2D substrates&#8212;an important setback since many biological processes, including tumor cell invasion and metastasis, and immune cell migration, involve 3D migration.
Direct observation chambers&#8212;where a chemoattractant solution is in contact with a 3D gel containing cells have also been also reported19,20. These chambers have two compartments, one containing a chemoattractant and one containing cells, are joined beside one another horizontally21 or as concentric rings22. These systems are pointed in the right direction, but do not keep a chemotaxis system for an extended period of time.
Furthermore, researchers have also examined diffusivity through collagen membranes in dialysis cells, as well as the diffusion of tracer molecules through collagen samples subjected to hydrostatic pressure23-25. Some diffusion experiments in collagen gels rely on physical and chemical modifications of the gel using magnetic fields and chemical incorporation26. A popular method for modeling diffusivity in collagenous tissues relies on the fluorescence imaging of continuous point photobleaching. This method has revealed anisotropy in the diffusion coefficients of macromolecules in oriented collagenous tissues. Yet, photobleaching has been used in articular cartilage and not collagen matrices. While similar, the necessary modeling experiments must be carried through specifically understanding the diffusion coefficient of collagen gels. More importantly, the systems do not utilize a method for measuring cell force generation.
Unfortunately, most systems seem to be missing one or two key elements for an ideal system: the allowing of cell tracking, a diffusion gradient understanding with a chemotactic factor through the matrix, a relatively simple set up with an ease of reproducibility, the minimization of cell-cell interactions, and the ability to measure dimensional units for quantification (i.e., velocity, force, specific concentration). Moghe et al.27 proposed a system that fulfilled most of these requirements in which cells were initially dispersed throughout the gel rather than concentrated on the filter surface, but was difficult to measure forces that the cell generates.
To this purpose, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays, which is based on time-lapse microscopy, coupled with image analysis techniques to measure cell forces in a 3D environment. This protocol provides a simple, yet innovative way of creating a simple 3D diffusion chamber that can be used to investigate 3D chemotaxis in different cells.

<table><tbody><tr><td>Silicone elastomer kit</td><td>Dow Corning Corp</td><td>182 SIL ELAST KIT .5KG</td><td>a two-part misture with a 10:1 mix</td></tr><tr><td>Live cell imaging chamber</td><td>Live Cell Instrument</td><td>CM-B18-1</td><td>CMB for 18&amp;nbsp;mm round coverslips</td></tr><tr><td>22&amp;nbsp;mm Glass Coverslip</td><td>Fisher Scientific</td><td>NC0180281</td><td>Neuvitro Corp. cover slip 22&amp;nbsp;mm 1.5</td></tr><tr><td>Machined aluminum metal cube</td><td></td><td></td><td></td></tr><tr><td>Hobby utility knife</td><td>X-Acto</td><td>X3201</td><td></td></tr><tr><td>3-(aminopropyl) trimethoxysilane</td><td>Sigma-Aldrich</td><td>281778-5ML</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Polysciences, Inc</td><td>00216A-10</td><td>Glutaraldehyde, EM Grade, 8%</td></tr><tr><td>50 ml tube</td><td>Fisher Scientific</td><td>14-432-22</td><td>Standard floor model and tabletop centrifuges</td></tr><tr><td>Glass Petri dish</td><td>Fisher Scientific</td><td>08-747A</td><td>Reusable Petri Dishes: Complete (60 x 15&amp;nbsp;mm)</td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>22-327-379</td><td>Fine Point Forceps</td></tr><tr><td>Cover glasses</td><td>Fisher Scientific</td><td>12-518-105A</td><td>Rectangle; 30 x 22&amp;nbsp;mm; Thickness No. 1</td></tr><tr><td>Tridecafluoro-1,1,2,2-tetrahydrooctyl</td><td>Gelest</td><td>SIT8174.0</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>320099</td><td>Acetic acid ACS reagent, &amp;ge;99.7%</td></tr><tr><td>Hexane</td><td>Sigma-Aldrich</td><td>296090</td><td>anhydrous, 95%</td></tr><tr><td>Ethyl alcohol</td><td>Sigma-Aldrich</td><td>E7023</td><td>200 proof, for molecular biology</td></tr><tr><td>High-precision diamond scribing tool</td><td>Lunzer</td><td>PV-081-3</td><td>Straight extended tip scribe, .020&quot; (.50&amp;nbsp;mm) diameter by .200&quot; (5.0&amp;nbsp;mm) tip length</td></tr><tr><td>Vacuum grease</td><td>Dow Corning</td><td>14-635-5C</td><td>High-Vacuum Grease</td></tr><tr><td>15 ml tube</td><td>Fisher Scientific</td><td>14-959-49D</td><td>15 ml conical centrifuge tubes with hydrophobic, biologically inert surface</td></tr><tr><td>10x phosphate buffered solution</td><td>Fisher Scientific</td><td>BP399-500</td><td>1.37 M Sodium Chloride, 0.027 M Potassium Chloride, and 0.119 M Phosphate Buffer</td></tr><tr><td>1&amp;nbsp;N sodium hydroxide</td><td>Sigma-Aldrich</td><td>38215</td><td>Sodium hydroxide concentrate</td></tr><tr><td>Collagen I, rat tail</td><td>BD Biosciences</td><td>354236</td><td>Rat tail&amp;nbsp;</td></tr><tr><td>Micro centrifuge tube</td><td>Fisher Scientific</td><td>02-681-332</td><td>Volume: 2.0 ml; &lt;br /&gt;O.D. x L: 13&amp;nbsp;x&amp;nbsp;40&amp;nbsp;mm; &lt;br /&gt;sterile; single-wrapped</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Carboxylate-modified microspheres</td><td>Invitrogen</td><td>&amp;nbsp;F-8813</td><td>Carboxylate-modified microspheres, 0.5 &amp;micro;m, yellow-green fluorescent (505/515), 2% solids</td></tr><tr><td>Rhodamine</td><td>Sigma-Aldrich</td><td>83689</td><td>Rhodamine B for fluorescence</td></tr></tbody></table>

planar gradient diffusion system to investigate chemotaxis in a 3d collagen matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

The ability of this assay to accurately assess the migration of the cell relies upon a good setup of the system. Therefore, it is critical for make sure to design the diffusion system mold accurately and take great care in placing both hydrophobic and hydrophilic coverslips, as illustrated in Figure 1. If the system is properly designed and during the diffusion modeling stage ensuring to find a very good linear starting line, one is able to achieve nice fluoresces images, as depicted in Figure 2, for analyzing diffusion of the system.
The key to a successful cell chemotaxis analysis is modeling the diffusion and solving the general diffusion equation to find the coefficient of diffusion and specific concentration equation through a computational software generated code. After importing the images into computational software and running analysis (Figure 3), the coefficient of diffusion can be assessed (Table 2). Figure 4 represents the plots of the diffusion of rhodamine through the five tested collagen concentrations. The shapes of the diffusion plot match the traditional shape of a linear diffusion model. It can be seen that diffusion of rhodamine is inversely proportional to the concentration of collagen&#8212;the lower the collagen density the faster the diffusion. This is to be expected, as this concentration of collagen has a higher porosity. The horizontal lines in the collagen diffusion plot at a concentration of 1.5 mg/ml (Figure 4B) are simply the result of over saturation of the tracer signal. More importantly, as shown in Figure 4 each line represents a specific point (x, y) inside the collagen, to whereas time increases the normalized concentration never exceeds the previous point at the same time (dt), indicating no reversal of the chemical. This is an important characteristic of a chemotaxis chamber to allow direct flow of a chemoattractant without reverse flow.
By examining the last ten images at each x location, one is able to generate a plot of the steady-state concentration of rhodamine at each of the locations. By extrapolating across the entire sample length (Figure 5), one can to look at the whole sample. This indicates that the linear diffusion gradient is consistent across the entire sample length, which can be utilized for cell experiments (Figure 6).
<img alt="Figure 1" src="/files/ftp_upload/52948/52948fig1.jpg" /> 
Figure 1: A schematic of planar gradient diffusion system mold assembly in live cell imaging chamber. (1) A computer rendition of the complete system consisting of (from bottom to top) a bottom housing (c) a hydrophilic 22 mm coverslip, mold, top housing, and a glass cover. (2) Dimensions of mold where (a) cut inserts are shown. (3) Mold with collagen matrix with (b) hydrophobic coverslips on each side slid into the cut inserts to create the 3-well system. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52948/52948fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52948/52948fig2.jpg" /> 
Figure 2: Example of rhodamine microscopy images in collagen. Representative images taken during rhodamine diffusion experiments, at same z-axis location, in the planar gradient diffusion system with a 1.0 mg/ml collagen concentration. Scale bar = 500 &#181;m where (a) is 0 min (b) 3 min (c) 7 min, and (d) 15 min after rhodamine was added to chamber. Arrow indicates direction of diffusion of rhodamine through the collagen matrix. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52948/52948fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52948/52948fig3.jpg" /> 
Figure 3: Example of analysis and summary of diffusion calculations generated by computational software code with a 2.5 mg/ml collagen concentration. (A) Black and white rendering of initial (dt = 0) image uploaded into computational software for analysis. (B) Specific points chosen by the user for intensity comparison and for diffusion calculations (x, y-axis are in pixels). (C) Diffusion profile of rhodamine throughout the collagen matrix where each line correlates to a red point in image B. (D) Concentration profile of rhodamine throughout the collagen matrix at steady-state. (E) Comparison between the smoothed data (blue dot) and true data from diffusion experiment (red dot) with difference between smoothed and true (green dot). (F) Normalized concentration of true (red dot) and fitted (green dot) data from computational software code throughout the experiment. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52948/52948fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52948/52948fig4.jpg" /> 
Figure 4: Example of diffusion profile through collagen matrix using the planar gradient diffusion system at different points. Each line indicates a different selected data points chosen from the computational software code by the user. Collagen matrix at a density of (A) 1.0 mg/ml (B) 1.5 mg/ml (C) 2.0 mg/ml (D) 2.2 mg/ml and (E) 2.5 mg/ml. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52948/52948fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52948/52948fig5.jpg" /> 
Figure 5: Example of a concentration (normalized) profile of rhodamine throughout the collagen matrix using the planar gradient diffusion system at steady-state. Circles are specific data points chosen though the computational software code by the user. Red line constructed using data points and extrapolating best line-fit (with R2 value shown) to show rhodamine concentration throughout the whole collagen matrix. Collagen matrix at a density of (A) 1.0 mg/ml (B) 1.5 mg/ml (C) 2.0 mg/ml (D) 2.2 mg/ml and (E) 2.5 mg/ml. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52948/52948fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52948/52948fig6.jpg" /> 
Figure 6: Example of cell migration experiments. A Z-slice microscope image of a neutrophil migrating in the 3D planar gradient system. Neutrophil migrates towards the chemoattractant gradient (white arrow depicts direction of gradient and the triangle shows its concentration profile), where the star indicates the initial position of the cell at the start of the experiment. Subplot shows cell in 3D space, where arrow indicates next time-point direction. Scale bar = 10 &#181;m. dt = 5 min between images (i.e., A-B). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52948/52948fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Collagen Cell Density</td>
			<td>PBS</td>
			<td>Fluorescent beads</td>
			<td>NaOH</td>
			<td>Collagen I Rat Tail</td>
			<td>Media</td>
		</tr>
		<tr>
			<td>2.5 mg/ml</td>
			<td>10%</td>
			<td>6%</td>
			<td>2%</td>
			<td>70.20%</td>
			<td>11.80%</td>
		</tr>
		<tr>
			<td>2.2 mg/ml</td>
			<td>10%</td>
			<td>6%</td>
			<td>2%</td>
			<td>61.70%</td>
			<td>20.30%</td>
		</tr>
		<tr>
			<td>2.0 mg/ml</td>
			<td>10%</td>
			<td>6%</td>
			<td>2%</td>
			<td>56.10%</td>
			<td>25.90%</td>
		</tr>
		<tr>
			<td>1.5 mg/ml</td>
			<td>10%</td>
			<td>6%</td>
			<td>2%</td>
			<td>42.10%</td>
			<td>39.90%</td>
		</tr>
		<tr>
			<td>1.0 mg/ml</td>
			<td>10%</td>
			<td>6%</td>
			<td>2%</td>
			<td>28.10%</td>
			<td>53.90%</td>
		</tr>
	</tbody>
</table>

Table 1: Custom collagen mixture adapted for the addition of fluorescent beads and cell viability.
<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr>
			<td>Concentration of collagen Gel</td>
			<td>Coefficient of Diffusion (cm2/sec)</td>
		</tr>
		<tr>
			<td>2.5 mg/ml</td>
			<td>1.03 x 10-6 &#177; 2.54 x 10-7</td>
		</tr>
		<tr>
			<td>2.2 mg/ml</td>
			<td>1.28 x 10-6 &#177; 3.53 x 10-7</td>
		</tr>
		<tr>
			<td>2.0 mg/ml</td>
			<td>6.48 x 10-6 &#177; 1.66 x 10-7</td>
		</tr>
		<tr>
			<td>1.5 mg/ml</td>
			<td>1.05 x 10-5 &#177; 2.04 x 10-7</td>
		</tr>
		<tr>
			<td>1.0 mg/ml</td>
			<td>1.39 x 10-5 &#177; 1.06 x 10-7</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>1.50 x 10-5&#160;</td>
		</tr>
	</tbody>
</table>
&#160;Table 2: Coefficients of diffusion.

Watch this Scientific Journal Video about Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix at JoVE.com

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

planar-gradient-diffusion-system-to-investigate-chemotaxis-3d

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.5K Views.  California State University, Long Beach. Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

Video: Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

Immunology and Infection

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Neuroscience

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

Summary

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Chapters in this video

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells