Danish Council for Independent Research, Technology and Production, grant #4005-00029

The experimental setup has no ethical and legal restriction and was conducted in agreement with Roskilde University. An overview of the experimental set-up can be seen in Figure 1 and the mechanisms of the optical camera in Figure 2.
1. Preparation
<ol>
	<li>Perform all steps in a sterile laminar flow-bench by cleaning the bench and all equipments with 70% ethanol prior to the start of the experiment.</li>
	<li>Place the optical camera in a conventional incubator at 37 &#176;C with 5% CO2 to ensure optimal conditions for the cells.</li>
	<li>Grow HaCaT cells in DMEM media with 10% FBS and 1x antibiotics (penicillin and streptomycin) in a T75 flask.</li>
	<li>Use 1 mL 1x trypsin to detach the cells and sub-cultivate them every 3-4 days, by dispersing it over the cellular layer. Pre-heat to room temperature before use.</li>
</ol>
2. Generating a Monolayer of HaCaT Cells in a 48-wells Microtiter Plate
<ol>
	<li>Remove the medium from the T75 flask with a Pasteur pipette.</li>
	<li>Wash the adherent cells in the flask by adding 5 mL 1x sterile PBS to the T75 flask. 
	Note: Ensure that the PBS is free of calcium and magnesium.</li>
	<li>Circulate the PBS and remove it with a Pasteur pipette.</li>
	<li>Repeat step 2.2-2.3.</li>
	<li>Add 1 mL 1x trypsin to the flask and disperse it across the cell layer.</li>
	<li>Incubate the flask at 37 &#176;C in a conventional incubator until the cells are detached (5-8 min is sufficient for most cell lines).</li>
	<li>View under the microscope if the cells are detached. Once the cells are detached, add 9 mL of the regular culture media with 10% FBS to inactivate the 1x trypsin.</li>
	<li>Count the cell with either a hemocytometer, a coulter-counter or similar. 
	Note: Trypan blue can be used to view dead cells. However, in 1x trypsin HaCaT cells are perfectly round when alive.</li>
	<li>Transfer 7.5 x 104 cells/well in 250 &#181;L 10% FBS media to a round-bottomed 48-well tissue culture plate (standard plate). 
	Note: Assay can also be set up in 6-, 12-, 24-, or 96-well plates, which all can be assayed under the optical microscope.</li>
	<li>Gently move the plate from side to side to ensure the cells are equally spread in the wells, or the cells will gather in the center of the wells. Inspect under a light microscope to make sure the cell is evenly spread in the media</li>
	<li>Put the plate in a conventional CO2 incubator at 37 &#176;C overnight or until the cells have adhered to the bottom of the wells.</li>
</ol>
3. Addition of Stimuli for Migration Effects
<ol>
	<li>Check if the wells are 90-95% confluent under a light microscope.</li>
	<li>Prepare 10 &#181;g/mL of mitomycin C in an appropriate amount of the medium. 
	Note: In this study, Mitomycin C is kept at 4 &#176;C in a stock solution at 1 mg/mL. Mitomycin C in solution can be stored up to one week in the dark at 2-8 &#176;C.</li>
	<li>Remove the spent media with a Pasteur pipette from each well.</li>
	<li>Add 250 &#181;L of 5 &#181;g/mL of mitomycin C to each well.</li>
	<li>Incubate the plate for 2 h at 37 &#176;C and 5% CO2.</li>
	<li>Prepare stimuli of peptides by diluting them in an appropriate amount of the medium. 
	Note: In this study, both the human and bovine Lactoferrin and Lactoferricin were tested at 25 &#181;g/mL in a volume of 250 &#181;L</li>
	<li>Remove the medium with mitomycin C with a Pasteur pipette.</li>
	<li>Wash the wells 1x with 250 &#181;L of 1x PBS (room temperature) and remove the wash with a Pasteur pipette.</li>
	<li>Add 250 &#181;L of 1x PBS and manually scratch the wells vertically with a 200 &#181;L pipette tip. Use a new pipette tip for each well.</li>
	<li>Remove the PBS and add 250 &#181;L peptide stimuli.</li>
	<li>Mount the plate in optical camera system by adjusting the screws on both sides of the plate. Leave the lid on the microtiter plate. 
	Note: The camera can operate with the lid open if necessary.</li>
</ol>
4. Set Up the Program for the Optical Camera on the Computer
<ol>
	<li>Click Acquire in the taskbar on the left.</li>
	<li>Choose the function Wound healing in the taskbar on the left.</li>
	<li>Choose the plate type by clicking below the Plate illustration.</li>
	<li>Unselect wells, not in use, by marking them and click Enable at the upper left side on the screen. 
	Note: All wells are selected by default.</li>
	<li>Adjust the focus of the lens on the wells to fit the position of the cells by right-clicking on one of the wells. 
	Note: Autofocus can also be adjusted manually by moving the bar to the right of the plate illustration.</li>
	<li>Set the time period for images under Picture Interval (e.g. (00:30:00)).</li>
	<li>Set the number of repetition to fit the desired period (e.g. 97 repetitions).</li>
	<li>Start the run by clicking Start button in the lower right corner.</li>
</ol>

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Biofilm+in+Wounds+as+an+Early+Indicator+for+Risk+for+Tissue+Infection+and+Wound+Chronicity.">Detection of Biofilm in Wounds as an Early Indicator for Risk for Tissue Infection and Wound Chronicity.</a> Annals of Plastic Surgery. 76 (1), 127-131 (2016).</li><li>Paul, C. 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S., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+cell-substrate+impedance+sensing+as+a+non-invasive+tool+for+cancer+cell+study.">Electrical cell-substrate impedance sensing as a non-invasive tool for cancer cell study.</a> The Analyst. 136 (2), 237-245 (2011).</li><li>Fredborg, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+optical+antimicrobial+susceptibility+testing.">Real-time optical antimicrobial susceptibility testing.</a> Journal of Clinical Microbiology. 51 (7), 2047-2053 (2013).</li><li>Jenssen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti+herpes+simplex+virus+activity+of+lactoferrin/lactoferricin+-+An+example+of+antiviral+activity+of+antimicrobial+protein/peptide.">Anti herpes simplex virus activity of lactoferrin/lactoferricin - An example of antiviral activity of antimicrobial protein/peptide.</a> Cellular and Molecular Life Sciences. 62 (24), 3002-3013 (2005).</li><li>Jenssen, H., Hancock, R. 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The authors have nothing to disclose.

The importance of migration in studies of development, wound healing, immunology, and cancer has led to several different methods to study migration. Migration can either be an expression of the expansion or the closure of the gap by the cells. Most often closure of the scratch is tested, as it is easier to grow cells in a monolayer and then make a scratch than growing the cells in a specific form8. Our study demonstrates an optimized method for manual scratching, as the technique is easy to master and low cost. We introduced the DNA synthesis inhibitor mitomycin C, an extra wash to optimize the scratch compared to published methods18, and the use of an optical camera that allows high-throughput studies.
The in vitro scratch assay requires an adherent cell line that binds properly to the bottom of the wells and to detect migration over a scratch, proliferation needs to be inhibited. These two features are essential for the assay. To detect true migration, several approaches have been described with lowering the serum concentration, starving the cell prior to scratching14, DMSO, or various inhibitors 13,27. In general, a serum-free media can be used, however, some cell lines, such as primary cell lines, need serum for the movement and the overall survival. Therefore, the choice should be made according to the cell line. Cell lines that require coating can be problematic in this assay, as manual scratches will disturb the coating as well.
Different cell types require the different number of cells to form a confluent monolayer depending on their size and growth rate. The HaCaT cells adhere after around 4-6 h but are left to settle overnight to make a stable monolayer. One of the major disadvantages of the scratch assay is irregular scratches8. In this study, we show that pre-washing with PBS and making the scratch in new PBS diminish accumulations of wounded and damaged cells along the edges of the scratch. This could plausibly be explained by the desmosomes junction connecting the cells in the skin. The junctions are Ca2+-dependent24 and by using PBS, the cations are removed, which could weaken the strong cell-cell adhesion. Thereby, the use of PBS could allow better removal of the cells individually, leading to less accumulation of the cells along the wound edge. Furthermore, the optical camera measures a wide part of the wells in all plate sizes and adapts to bents in the scratch via its three-step algorithm. Thereby, by pre-washing with PBS and by using the optical camera, the disadvantages of the manual scratches are eliminated. The z-stacking of images makes focusing on the cells more precise, but depending on how different cell types grow, the focus can be a problem. If the cells grow too much, the focus of the camera will shift from in-focus to out-of-focus. Thus, if mitomycin C is not used, it can be advantageous to set the focus a little higher if your experimental setup will run for more than 24 h. The optical camera has an easy set-up that is operated through a standard computer with an intuitive software that can measure plates from 6- to 96-well microtiter plates and therefore enables various test set-ups. When choosing the number of images to be taken, few limitations exist. If a 96-well plate is used, the number of pictures one can obtain is only limited to space on the computer, as data-sets can become large, and by the speed of the camera. If many pictures are required in a quick succession, the movement of the camera can be a limiting factor, as it needs time to scan each well.
The optical system enables simple but powerful monitoring. The pictures of the scratch are analyzed in regard to the process of the closure of the gap but can also be used for morphological characterization of the cells. The software marks the cells yellow and leaves the background color grey as a standard feature, to better visualize the wound edge, though raw images obviously are available should that be needed for e.g. publications. The HaCaT cells have strong cell-cell interactions which make the migration occur in a dynamic sheet28. This feature makes it easy to see lamellipodia of the migrating cells over time (Figure 4).
This study demonstrates the effect of the proteins Lactoferrin and their derived N-terminal peptides Lactoferricin, and EGF as a positive control, but could be modified to test different drugs, peptides/proteins, and combinations, as inhibitors or stimulators of wound healing. The scratch assay can be altered and optimized to fit various cell lines and compounds that can affect migration. Together, with its low cost and easy manageability, this assay is very accessible for most laboratories. The optical camera could be used to test different drugs, inhibitors or stimulators for migration, but also proliferation.

Migration is an important process that influences several physiological aspects. In wound healing, migration facilitates re-epithelization of the skin. In non-healing wounds, such as chronic wounds, opportunistic bacteria cause infection of the wound, and open wounds are ideal for bacteria growth and formation of biofilm1,2. Biofilm is one of the causes of the development of resistant bacteria, which is one of the biggest threats to our modern society3. In wound healing, the immune cells cannot penetrate the biofilm. In cancer, migration of the cancer cells is a pivotal step of metastasis, which is the primary cause of death for patients with solid tumors4,5. Therefore, it is crucial to be able to study migration.
The scratch assay is often used to test cell migration because it is cheap and easy to perform on adherent cell lines, such as fibroblast, endothelial, and epithelial cell lines6,7. Today, several methods exist for performing scratches8, and different materials have been reported to be used, including toothpicks9, cell scrapers10, lasers11, and electric currents12. All of the methods have different pros and cons, and the method should be decided upon according to the cell type, budget, and analysis tool. As an example, if the used cell type requires coating, manual pressure removal, e.g. with a toothpick or pipette tip, will influence migration within the area, a different method should be used. In this study, we will focus on manual scraping.
A downside for manual scraping is the variation of the size, shapes of the scratches, and accumulation of cell on the edges of the scratch. Many protocols exist, and a highly cited paper advises increased speed and/or thorough washing after scratching13. Additionally, several methods have been used to minimize proliferation, since the proliferation of the cells cannot be distinguished from migration and therefore may give false-positive results of migration. Some reported methods are serum starvation preceding the scratch14 and decreasing the amount of serum15. However, neither of these methods can ensure that there is no proliferation. Therefore, we report a pre-treatment of the cells with mitomycin C to ensure true results of migration. Mitomycin C is an antibiotic that inhibits DNA synthesis by forming a covalent bond with the DNA, which prevents the DNA from separating16. By pre-treating with mitomycin C and washing with PBS before scratching, the detected closure of the gap demonstrates the true migration of the cells (Figure 1).
A characteristic of all the methods is the need for a system to analyze the process of migration17. A common and an inexpensive method to analyze the progress is to use a light-field microscope to take images of the scratch at pre-determined time points, followed by an analysis of the closure of the gap in an image software18,19. This method is time-consuming, unreliable with regards to taking pictures at the same spot and focus, and the movement from incubator to camera forces cycles of temperature changes while pictures are taken. Other methods have been developed to detect migration by measuring the current flow. The current changes, as cells cover more space on the plate, which is read as an increase in the impedance20. By measuring impedance, the environment of the cells is not affected, and the method is therefore considered non-invasive. This method relies on specialized plates with gold electrodes, which are expensive, but they enable detection of many processes, such as cellular adherence, proliferation, migration, and cell death. A big disadvantage of this method is that the impedance measurement does not directly indicate migration, as factors such as temperature have a great impact on the impedance. Changes in the impedance can be caused by several factors that are not reviewed by the software, making the analysis of the data more difficult. Therefore, the lack of visualization requires a conventional light microscope to verify migration.
To overcome many of the downsides of the available techniques, we use a real-time automated optical camera. The optical camera is a detection system with a high-throughput microscope in a small platform with a lens, illumination unit, and a digital camera. It has a built-in software, which can test migration and proliferation among other things. To detect migration, two algorithms are used to analyze the growth and the migration kinetics of cells, which are called the proliferation and the wound healing analysis, respectively. The proliferation analysis is based on a two-step image segmentation algorithm that applies texture analysis to detect the difference between cell-covered areas (shown in yellow) and empty background areas by advanced histogram analysis. The wound healing analysis is based on a three-step algorithm that detects the cell monolayer, locates the wound gap, and determines the gap width. These steps are conducted at user-determined time-points, and the data is presented as covered area, gap width, and gap area. One of the great advantages of this machine is that it enables imaging of adherent cells through liquid because of the angle of the camera21. The camera lenstakes tilted images that are projected to a z-stack of a single z-plane generate to ensure that the pictures are in-focus (Figure 2). z-stacks are generated by merging the series of pictures (e.g. 40) taken perpendicular to the wound edge, and across the entire wound. The z-stacks enables capturing of the depth of the cell layer as well as an in-focus plane across the wound. Furthermore, the series of images can be put together to a video collection of the images, with the click of a button.
We demonstrate an optimized protocol for detection of migration in the keratinocyte cell line HaCaT. We tested Lactoferrin and its N-terminal peptide, Lactoferricin, from humans and cows, to analyze their effect on migration as these molecules have been demonstrated to have numerous applications as antimicrobial and immune modulating molecules22,23. The four compounds were compared to untreated HaCaT cells and epidermal growth factor (EGF) was used as a positive control.

<table border="0" cellpadding="0" cellspacing="0" width="712">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">oCelloScope</td>
			<td style="width:156px;">BioSense Solution ApS</td>
			<td style="width:119px;"></td>
			<td style="width:221px;">Optical camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UniExplorer software&#160;</td>
			<td style="width:156px;">BioSense Solution ApS</td>
			<td style="width:119px;"></td>
			<td>(8.0.0.7144 (RL3))</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HaCaT cell</td>
			<td style="width:156px;"></td>
			<td style="width:119px;"></td>
			<td>Donated from Bispebjerg Hospital</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM (1x) + GlutaMAX</td>
			<td style="width:156px;">Gibco</td>
			<td style="width:119px;">31966-021</td>
			<td>Medium for HaCaT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:156px;">Gibco</td>
			<td style="width:119px;">10270</td>
			<td>Fetal bovine serum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:156px;">Gibco</td>
			<td style="width:119px;">D837</td>
			<td>Without Mg+ and Ca2+</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pencillin-Streptomycin</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">P0781</td>
			<td>Antibiotics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin (10x)</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">T3924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mitomycin C</td>
			<td style="width:156px;">Tocris</td>
			<td style="width:119px;">3258</td>
			<td>Inhibits proliferation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microtiter plate</td>
			<td style="width:156px;">Greiner Bio-one</td>
			<td style="width:119px;">677 180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator</td>
			<td style="width:156px;">Panasonic</td>
			<td style="width:119px;"></td>
			<td>37&#176;C, 5% CO2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer</td>
			<td style="width:156px;">Assistent</td>
			<td style="width:119px;"></td>
			<td>T&#252;rk (0.1 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipet boy</td>
			<td style="width:156px;">Accu-Jet pro</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized scratch assay for in vitro testing of cell migration with an automated optical camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

HaCaT is a keratinocyte cell line, one of the most abundant cell types in the skin. When a wound occurs, the skin cells start to proliferate and migrate over to the wound bed to close the wound (Figure 3). Both processes are, therefore, of utmost importance for proper healing.
The optical camera can follow both processes in real-time. For migration, the system gives three data sets: cell covered area, gap width, and gap area. When monitoring scratches treated with Lactoferrin, Lactoferricin or EGF, the optical microscope allows us to monitor these three parameters over time. After 48 h incubation, we observe that EGF has resulted in 95% closure of the wound, compared to 38% closure of the untreated control (Figure 3A). The different versions of Lactoferrin and Lactoferricin are placed between 33% and 62% wound closure with the peptides being more potent than the parent proteins. The percentage of wound closure, determined from changes in gap width from the initial scratch, can also be plotted (Figure 3B).
HaCaT cells have a strong cell-cell interaction and when the cells migrate, series of steps are required24. Shortly, the leading cells disrupt their site of adhesion, protrude, and contract the cytoskeleton to move25. The protrusion of the leading cells can easily be viewed with the optical camera (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57691/57691fig1.jpg" /> 
Figure 1: Schematic flowchart of the scratch assay to test migration on an optical camera. HaCaT cells are plated on a 48-well plate and allowed to adhere for 24 h. The cells are pre-treated with mitomycin C for 2 h to inhibit proliferation, and the scratch is performed and followed by the addition of peptide stimuli. The plate is inserted into the unit of the optical camera, and migration is followed for 48 h.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57691/57691fig2.jpg" /> 
Figure 2: The scanning technology of the optical camera. The optical lens unit of the automated optical camera is angled at 6.25&#176; to the horizontal plane to enable scanning through various solutions such as bacterial and cellular solutions. The acquired series of images contain image stacks of all focuses, both out-of-focus and in-focus, to ensure images with the cells in-focus. Modified from26.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57691/57691fig3.jpg" /> 
Figure 3: Migration of pre-treated mitomycin C HaCaT cell over 48 hours. (A) Migration is detected on the automated optical camera every half hour for 48 h. The cells are pre-treated with mitomycin C to inhibit proliferation. The cells are treated with 25 &#181;g/mL of either humane- or bovine-Lactoferrin (H-LF or B-LF) or -Lactoferricin (H-LFcin, B-LFcin). Epithelial growth factor (EGF) is used as a positive control at 500 ng/mL. Untreated HaCaT cells are used as a control. (B) Graphic illustration of migration of the treatments. The results are representatives of three independent runs, and results are given as a percentage closure of the initial scratch *p&#60;0.05, **p&#60;0.01, ***p&#60;0.001.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57691/57691fig4.jpg" /> 
Figure 4: Protruding lamellipodia of treated HaCaT cells. Lamellipodia of the HaCaT cells is marked by the black arrows. The strong cell-cell interaction of the HaCaT results in dynamic movement of the cells as they migrate and can easily be observed over time.

Watch this Scientific Journal Video about Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera at JoVE.com

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...

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Universidad San Sebastian

Research

JoVE Journal

Biology

22.8K Views.  Roskilde University. Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.

Video: Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Preparation of Cell Extracts by Cryogrinding in an Automated Freezer Mill

The ease of genetic manipulation and the strong evolutionary conservation of eukaryotic cellular machinery in the budding yeast Saccharomyces cerevisiae has made it a pre-eminent genetic model organism. However, since efficient protein isolation depends upon optimal disruption of cells, the use of yeast for biochemical analysis of cellular proteins is hampered by its cell wall which is expensive to digest enzymatically (using lyticase or zymolyase), and difficult to disrupt mechanically (using a traditional bead beater, a French press or a coffee grinder) without causing heating of samples, which in turn causes protein denaturation and degradation. Although manual grinding of yeast cells under liquid nitrogen (LN2) using a mortar and pestle avoids overheating of samples, it is labor intensive and subject to variability in cell lysis between operators. For many years, we have been successfully preparing high quality yeast extracts using cryogrinding of cells in an automated freezer mill. The temperature of -196 &#176;C achieved with the use of LN2 protects the biological material from degradation by proteases and nucleases, allowing the retrieval of intact proteins, nucleic acids and other macromolecules. Here we describe this technique in detail for budding yeast cells which involves first freezing a suspension of cells in a lysis buffer through its dropwise addition into LN2 to generate frozen droplets of cells known as "popcorn". This popcorn is then pulverized under LN2 in a freezer mill to generate a frozen "powdered" extract which is thawed slowly and clarified by centrifugation to remove insoluble debris. The resulting extracts are ready for downstream applications, such as protein or nucleic acid purification, proteomic analyses, or co-immunoprecipitation studies. This technique is widely applicable for cell extract preparation from a variety of microorganisms, plant and animal tissues, marine specimens including corals, as well as isolating DNA/RNA from forensic and permafrost fossil specimens.

We describe a reliable method for the preparation of whole cell extracts from yeast or other cells using a cryogenic freezer mill that minimizes degradation and denaturation of proteins. The cell extracts are suitable for purification of functional protein complexes, proteomic analyses, co-immunoprecipitation studies and detection of labile protein modifications.

The ease of genetic manipulation and the strong evolutionary conservation of eukaryotic cellular machinery in the budding yeast Saccharomyces cerevisiae ...

A Cost Effective and Adaptable Scratch Migration Assay

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as development and mounting an immune response. When a defect or alteration occurs with the cell migration process, it can have detrimental outcomes (i.e., cancer metastasis, wound healing, and scar formation). Due to the importance of cell migration, it is necessary to have access to a cell migration assay that is affordable, adaptable, and repeatable. Utilizing the common scratch migration assay, we have developed a new approach to analyzing cell migration that uses general laboratory equipment. The method described uses visual markers that allow for recapturing specific areas of interest without the use of time-lapse microscopy. In addition, it provides flexibility in the experimental design, ranging from altering the migration matrix substrate to the addition of pharmacological modifiers. Furthermore, this protocol outlines a way to account for the area of cell migration, which is not considered by several methods when examining cell migration. This new approach offers a scratch migration assay to a larger audience and will provide greater opportunity for researchers to examine the physiological and pathophysiological impact of cell migration.

We present a cost-effective method to the scratch migration assay that provides a new approach for determining cell migration without the use of equipment-intensive methods. While fibroblasts were used in this protocol, it can be adapted and utilized to study additional cell types and influences on cell migration.

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.