This work was supported by funding from Sapienza University of Rome and the Italian Cystic Fibrosis Research Foundation (Project FFC#11/2014 adopted by FFC Delegations from Siena, Sondrio Valchiavenna, Cerea Il Sorriso di Jenny, and Pavia). Part of this work was also supported by FILAS Grant Prot. FILAS-RU-2014-1020. 
We are grateful to Dr. Loretta Ferrera (U.O.C. Genetica Medica, Istituto Giannina Gaslini, Genova, Italy) for providing the bronchial epithelial cells.

1. Cell Preparation
<ol>
	<li>Seed 2.5x106 cells in 10 mL of Minimum Essential Medium (MEM) supplemented with 2 mM glutamine (MEMg), plus 10% fetal bovine serum (FBS), antibiotics (0.1 mg/mL of penicillin and streptomycin), and puromycin (0.5 &#181;g/mL for selection and maintenance of the cell line) in a T75 flask. Incubate the flask at 37 &#176;C and 5% CO2 for 2 days. Before starting the experiment, check the confluence of cells under an inverted microscope. 
	NOTE: The cells used for the experiment are immortalized human bronchial epithelial cells transduced with a lentiviral vector conferring resistance to puromycin. They stably express a functional CFTR14,15.</li>
	<li>Once the cells&#39; confluence has reached 90-100%, aspirate the medium from the flask and discard it into a waste bottle under a biological safety cabinet class II. Wash the cells with 6 mL of phosphate buffered saline without calcium and magnesium (CMF-PBS). Gently rock the flask manually and discard CMF-PBS. 
	NOTE: Be careful not to touch the cell monolayer with the pipette.</li>
	<li>Add 10 mL of CMF-PBS and incubate the flask at 37 &#176;C and 5% CO2 for 10 min.</li>
	<li>Aspirate CMF-PBS and discard it. Then add 2 mL of trypsin/EDTA to the flask.</li>
	<li>Gently rock the flask, allowing the solution to completely coat the cells, and incubate the flask at 37 &#176;C and 5% CO2 for 10 min until the cells are visibly detached under a microscope. 
	NOTE: At the end of the incubation time, the cells should appear rounded and not attached to the plastic surface. If the cells are not well detached, manual agitation may be necessary.</li>
	<li>Add 10 mL of MEMg plus 10% FBS to inactivate trypsin and collect the cells by washing the bottom of the flask. Transfer the volume into a conical 50 mL tube.</li>
	<li>Centrifuge the tube for 5 min at 80 x g.</li>
	<li>Aspirate the supernatant and re-suspend the cells in 6 mL of MEMg plus 10% FBS. Pipette repeatedly to break up any clumps.</li>
	<li>Take out 10 &#181;L of cell suspension with a micropipette and inject the volume under the cover glass previously put over a Burker or Neubauer chamber.</li>
	<li>Count the cell number.</li>
</ol>
2. Cell Seeding in the Culture Inserts
<ol>
	<li>In each well of a 12-well plate, transfer the culture insert with sterile tweezers (Figure 1). Use tweezers to press along the inserts edges in order to fix them to the surface of the plate. 
	NOTE: Inserts have a sticky underside that allows adhesion.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56825/56825fig1.jpg" /> 
Figure 1: Schematic representation of the silicone culture inserts, properly put into wells of a 12-well plate. <a href="//cloudfront.jove.com/files/ftp_upload/56825/56825fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Properly dilute the cell suspension in MEMg plus 10% FBS. Fill each compartment of the insert with 70 &#181;L of cell suspension (about 3.5x104 cells/chamber). 
	NOTE: The density of cells applied in each compartment depends on the type of cells. It is recommended to use a cell density that leads to complete confluence within 24 h.</li>
	<li>Under the microscope, check that cells are not leaking from the insert compartments and incubate the 12-well plate for 24 h at 37 &#176;C and 5% CO2.</li>
</ol>
3. Pseudo-Wound Healing Assay
<ol>
	<li>After incubation, visualize the cells under the inverted microscope to verify that confluent cell monolayers have been formed.</li>
	<li>Re-suspend the test compounds (e.g., AMPs) in 1 mL of MEMg. 
	NOTE: Prepare fresh AMPs dilutions starting from the stock solution stored at -20 &#176;C.</li>
	<li>Gently remove the inserts by sterile tweezers. Be careful not to break the cell monolayers. Transfer the inserts onto absorbent paper. 
	NOTE:&#160;To re-use the same inserts, sterilize them in 70% ethanol for at least 3 h. It is recommended to throw them away afterwards.</li>
	<li>To remove non-adherent cells, add 1 mL of MEMg per well using a micropipette. Close the plate and gently rock it. 
	NOTE: Do not add medium directly on top of cell monolayers to avoid their disruption.</li>
	<li>Aspirate the medium and replace it with 1 mL of MEMg per well. Close the plate and visualize the cell-free gaps (created by the inserts) under the inverted microscope at 4X magnification, equipped with a video-camera. Acquire images at time zero (T0) and save them in a .jpg format.</li>
	<li>Remove the medium from the wells, wash them with 1 mL of PBS, and discard it.</li>
	<li>Add the test compounds (prepared at point 3.2) to the wells. For untreated control samples, add 1 mL of MEMg. Incubate the plate at 37 &#176;C and 5% CO2.</li>
	<li>After 15, 20, and 24 h treatment, observe the cell migration under the microscope at 4X magnification and acquire images. 
	NOTE: During this step, try to capture images in the same areas as for T0. The choice of time intervals at which images are captured depends on the cell migration speed.</li>
	<li>To study the effect of some selective inhibitors, i.e. AG1478, on cell migration, aspirate the medium from each insert compartment before removing inserts.
	<ol>
		<li>Wash each compartment with fresh MEMg and fill it with 70 &#181;L of MEMg supplemented with AG1478.</li>
		<li>After 30 min of incubation at 37 &#176;C and 5% CO2, proceed as described from point 3.3. 
		NOTE: During the washing step and pretreatment of cell monolayers with the specific inhibitors, be careful not to remove the inserts.</li>
	</ol>
	</li>
</ol>
4. Image Analysis
<ol>
	<li>Upon completion of the experiment, select images of the most representative samples of the various experimental groups, and create a zip file containing single images at T0, T15, T20, and T24 h. 
	NOTE: Single images are taken at the selected time intervals for all samples. Run triplicates for each experiment, which is repeated at least three times. At the end, for all experimental groups, a minimum of 3 images (&#34;a&#34;, &#34;b&#34;, &#34;c&#34;, etc., deriving from each independent experiment) at each time point are analyzed.</li>
	<li>Upload the zip file into the image analysis software which automatically provides by e-mail a spreadsheet summary file containing the experimental data of cell-covered and scratch areas (as percentage) at the selected time points. 
	NOTE: The recognition of the leading edge and the gap area is largely based on edge detection method (aimed at identifying points at which the image brightness sharply changes).</li>
	<li>Save the data and collect them. Normalize all data with respect to the mean value at time zero. Calculate the average value of the normalized data of all replicates at each time point and the relative standard error (SEM). By using two-way analysis of variance (ANOVA), perform statistical analysis with a proper statistics software. Differences between peptide-treated groups and control groups at different time intervals are considered to be statistically signi&#64257;cant for a p&#60;0.05.</li>
	<li>Plot the obtained data into a graph as a histogram, which shows the percentage of cell-covered area of all sample groups versus the selected time intervals.</li>
</ol>

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The authors have nothing to disclose

Cell migration is an essential process in many physiological and pathological events including wound healing, embryonic development, and cancer metastasis. The basic procedure to study cell migration in vitro involves: (i) the creation of a cell monolayer, (ii) the production of a pseudo-wound in the confluent layer of cells, (iii) the capture of images at different time intervals until wound closure is reached, and (iv) the analysis of the image sequence in order to quantify the migration speed of the chosen ce...

It is largely known that wound healing in animals is a fundamental process to re-establish the integrity and normal function of tissue layers after injury1. Despite epithelial surfaces exposed to the external environment (e.g., the skin, respiratory, and gastrointestinal tracts) form&#160;a protective barrier from physical and chemical insults, the formation of wounds can easily occur, especially after surgery or microbial infections2. As an example, colonization of lung tissue by the opportunistic bacterial pathogen Pseudomonas aeruginosa, especially in cystic fibrosis (CF) sufferers, leads to damage of the airways epithelium with consequent respiratory failure3,4. Wound healing is a complex host repair mechanism to restore the normal architecture of an injured tissue5. It is characterized by initial inflammation, followed by a regeneration period encompassing epithelialization, angiogenesis, and tissue remodeling with collagen production and cell differentiation6,7,8. To ensure epithelial integrity and to control microbial proliferation, all living organisms produce defense molecules, including antimicrobial peptides (AMPs)9,10. The wound healing process is very difficult to simulate in vitro due to the lack of cell debris and complex interactions among different cell types. However, the in vitro ability of a peptide to accelerate the closure of a pseudo-wound by stimulating migration of epithelial cells is indicative of its ability to heal a compromised epithelium. Indeed, cell migration is a rate-limiting event in wound healing, and studying factors that can affect cell migration will help to target therapies for improved wound healing.
Here, a highly reproducible experimental assay is provided based on special silicone culture inserts to evaluate cell migration in vitro. It is based on the creation of a 500 &#956;m gap (pseudo-wound) on a confluent cell monolayer. The cells at the edge of the artificial &#34;wounded&#34; field will start migrating into the cell-free area, forming new cell-cell contacts. The culture insert represents a new tool for fast wound healing experiments. Two reservoirs separated by a 500 &#956;m wall are provided, and they can be properly placed into a 3-cm dish plate or in the well of a 12-well plate. Filling each compartment of the insert with a cell suspension allows cells to grow in each designated area until confluence, while removal of the insert will engender a clean cell-free gap of approximately 500 &#956;m (the same width as the separation wall). A proper cell culture medium supplemented with a test compound can then be added into the dish plate/well. Afterwards, the gap closure can be visualized at different time intervals under an inverted microscope, preferably one equipped with a video-camera for image acquisition. Finally, measurement of changes in the cell-covered area by the web-based automated image analysis program will allow the quantification of the speed of wound closure and cell migration. Overall, this method is a step forward with respect to the classical assay, where a scratch is manually made by incising confluent cell monolayers with a sterile needle or a pipette tip11. Indeed, the last procedure can destroy the plastic bottom of the dish plate/well and the surface coating, creating wrinkles. In addition, the &#34;wounded&#34; area does not have a well-defined width along the entire length of the gap, as this highly depends on the pressure applied by researchers to the needle/tip. Furthermore, the dislodged cells can form clumps of living and dead cells at the edges of the scratch; moreover, the spreading of living cells into the &#34;wounded&#34; area can interfere with the velocity of cell migration, generating non-reproducible results12.
In addition, thanks to a scratch image analysis platform, users can rapidly receive (within minutes) quantitative data on the migratory behavior of the selected cells without the necessity of acquiring additional software. This platform is capable of analyzing phase contrast microscopy images of low (~5X), medium (~10X), and high (~20X) magnification. After uploading a zip file of images (in *.jpg, *.jpeg, *.jp2, *.png, *.gif, *.tiff format) the analysis is automatically conducted to generate a summary file that shows the percentage of both cell-covered areas and scratch areas, as well as the speed of cell migration, at distinct time intervals.
In this work, by using a frog-skin AMP-derivative, i.e. Esc(1-21) and its diastereomer Esc(1-21)-1c13, and a bronchial cell line expressing the functional CF transmembrane conductance regulator (CFTR)14,15, an example of peptide-induced cell migration in comparison with untreated (control) samples is provided. Note that the airway epithelium and CFTR play a crucial role in maintaining lung function and wound repair16. Furthermore, by means of selective inhibitors (e.g., AG1478)17 of the epidermal growth factor receptor (EGFR), evidence that migration of bronchial cells induced by the aforementioned peptides involves activation of EGFR12,18 is reported.
In summary, the goal of this procedure is to show how the usage of such silicone culture inserts represents a fast and easily accessible assay to accurately determine migration of adherent cells (e.g., bronchial epithelial cells) and the molecular mechanisms controlling such events.

<table border="0" cellpadding="0" cellspacing="0" width="852">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Minimal essential medium (MEM)&#160;</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB2071L</td>
			<td style="width:256px;">Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Glutamine</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3000D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Heat inactivated Fetal Bovine Serum (FBS)</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECS0180DH&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin and Streptomycin</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3001D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Puromycin</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">P8833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Trypsin/EDTA 1X in PBS</td>
			<td style="width:179px;">Euroclone</td>
			<td style="width:119px;">ECB3052D</td>
			<td>Warm at room temperature before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">DPBS without calcium and magnesium (CMF-PBS)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DPBS with calcium and magnesium (PBS)</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ibidi Culture-Insert 2 well</td>
			<td style="width:179px;">Ibidi&#160;</td>
			<td style="width:119px;">80209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Wimasis Image Analysis</td>
			<td style="width:179px;">Ibidi&#160;</td>
			<td style="width:119px;">30002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PRISM software&#160;</td>
			<td>GraphPad</td>
			<td style="width:119px;">version 6.0</td>
			<td></td>
		</tr>
	</tbody>
</table>

a novel in vitro wound healing assay to evaluate cell migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

This protocol was used to determine the wound healing effect of Esc(1-21) and Esc(1-21)-1c in terms of cell migration activity induced on bronchial epithelial cells expressing the functional CFTR. In this assay, culture inserts were placed in wells of a 12-well plate, and each compartment was seeded with 35,000 cells in MEMg supplemented with 10% FBS. The cells reached complete confluence within 24 h. Afterwards, a 500 &#956;m gap was generated, and each well was filled with MEMg containi...

Watch this Scientific Journal Video about A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration at JoVE.com

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

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21.6K Views.  Sapienza University of Rome. Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

Video: A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

A CFSE-based Assay to Study the Migration of Murine Skin Dendritic Cells into Draining Lymph Nodes During Infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph node (DLN). The mobilization of DCs to the DLN is complex and remains to be fully elucidated during infection. Herein described is the use of an innovative, simple assay that relies on the fluorochrome 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) to track the migration of DCs during footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin (BCG) in C57BL/6 mice. This assay enables the characterization of skin DC sub-populations that actively relocate to the draining, popliteal LN in response to BCG. This protocol originates from a BCG model where migratory skin DCs were identified by flow cytometry. The assay is amiable to the study and identification of DCs or other cells that home to the popliteal LN after inoculation of microbes, their metabolites or other inflammatory stimuli in the footpad, and consequently to study factors that regulate the migration of these cells.

An assay in mice to track cell migration from skin to draining lymph node is described which enables the characterization of skin Dendritic cells mobilized to the lymph node after footpad infection with Bacille Calmette-Gu&#233;rin.

Dendritic cells (DCs) are important for initiating immune responses, in part through their ability to acquire and shuttle antigen to the draining lymph ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor microenvironment and the underlying mechanism is still largely unknown. The lack of a consistent and reliable source of NK cells limits the research progress of NK cell immunity. Here, we report an in vitro system that can provide high quality and quantity of bone marrow-derived murine NK cells under a feeder-free condition. More importantly, we also demonstrate that siRNA-mediated gene silencing successfully inhibits the E4bp4-dependent NK cell maturation by using this system. Thus, this novel in vitro NK cell differentiating system is a biomaterial solution for immunity research.

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Here, we present a protocol to mass-produce gene-silencing murine NK cells by using a feeder-free differentiation system for mechanistic study in vitro and in vivo.

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...