Supported by NIEHS grants #R01 ES013268-01A2, and the contents are solely the responsibility of the author and do not necessarily represent the official views of the NIEHS, and supported by CETOCOEN UPgrade project No. CZ.1.05/2.1.00/19.0382.

The protocol for this study was approved by the Animal Care and Utilization Committee of the National Institutes of Health Sciences of Japan, which is where the in vivo experiments were done, to assure that the rats were treated humanely and with regard for alleviation of suffering.
1. SL-DT Bioassay
<ol>
	<li>Seed 3 x 105 WB-F344 rat liver epithelial cells onto 35 mm diameter culture plates containing Eagles modified medium plus 5% fetal bovine serum, and culture the cells in an incubator at 37 &#186;C, 100% relative humidity (RH), 5% CO2.</li>
	<li>Culture the cells until they reach 100% confluence (typically, 2 days) and conduct the desired experimental treatment of the cells as described below.
	<ol>
		<li>Conduct Dose Response Experiments
		<ol>
			<li>Add 10 &#181;l and 20 &#181;l of a 2 mM stock solution of phenanthrene in 100 % acetonitrile and 4 &#181;l, 6 &#181;l, and 8 &#181;l of a 20 mM stock solution of phenanthrene in 100 % acetonitrile to three plates of cells containing 2 ml of growth medium for each added volume of stock solution.</li>
			<li>Add 4 &#181;l, 6 &#181;l, 8 &#181;l, 10 &#181;l, and 20 &#181;l of a 100% solution of acetonitrile to three plates of cells containing 2 ml of growth medium for each added volume of acetonitrile solution to serve as the vehicle control.</li>
			<li>Incubate plates for 15 min in an incubator set at 37 &#176;C, 100% RH and 5% CO2.</li>
			<li>Proceed to step 1.3.</li>
		</ol>
		</li>
		<li>Conduct Time Response Experiments
		<ol>
			<li>Add 7 &#181;l of a 20 mM stock solution of phenanthrene in 100% acetonitrile to three plates of cells containing 2 ml of growth medium for each time point (i.e., 0, 1, 2, 3, 4, 5, 10 min).</li>
			<li>Add 7 &#181;l of 100% acetonitrile to three plates of cells containing 2 ml of growth medium for each time point to serve as the vehicle control.</li>
			<li>Incubate plates for each designated time point in an incubator at 37 &#176;C, 100% RH and 5% CO2.</li>
			<li>Proceed to step 1.3.</li>
		</ol>
		</li>
		<li>Conduct Time Recovery Experiments
		<ol>
			<li>Add 7 &#181;l of a 20 mM stock solution of phenanthrene in 100% acetonitrile to three plates of cells containing 2 ml of growth medium for 15 min.</li>
			<li>Add 7 &#181;l of 100% acetonitrile to three plates of cells containing 2 ml of growth medium for 15 min to serve as the vehicle control.</li>
			<li>Decant the medium containing either the phenanthrene or acetonitrile and rinse 3x each with 3 ml of phosphate buffered saline (PBS).</li>
			<li>Add 2 ml of fresh growth medium to each plate and incubate for the desired recovery times (i.e., 25, 35, 45, 60, 90, 120, 150, 180, 240, 360 min) in an incubator at 37 &#176;C, 100% RH and 5% CO2.</li>
			<li>Proceed to step 1.3.</li>
		</ol>
		</li>
		<li>Conduct Mechanism Experiments
		<ol>
			<li>Add the signal transduction inhibitor, i.e., 5 &#181;l of a 20 mM stock solution of D609 in 100% acetonitrile to three plates of cells containing 2 ml of growth medium for 15 min.</li>
			<li>Add 5 &#181;l of 100% acetonitrile to three plates of cells containing 2 ml of growth medium for 15 min to serve as the vehicle control.</li>
			<li>Add 7 &#181;l of a 20 mM stock solution of phenanthrene in 100% acetonitrile to three plates of cells containing 2 ml of growth medium plus the D609 for an additional 15 min.</li>
			<li>Add 7 &#181;l of 100% acetonitrile to three plates of cells containing 2 ml of growth medium plus the 100% acetonitrile for an additional 15 min to serve as the vehicle control.</li>
			<li>Proceed to step 1.3.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Discard the culture medium by either gently pouring off the medium or by vacuum suction. 
	Note: Dispose culture medium containing hazardous waste appropriately.</li>
	<li>Rinse the cells three times each with 3 ml of PBS and either decant or aspirate between rinses.</li>
	<li>Pipette 1 ml of 1 mg/ml Lucifer Yellow dye dissolved in PBS (LY) into each cell plate.</li>
	<li>Preload the dye into the cells by gently rolling a #20 surgical steel blade with a rounded edge through a population of cells in three different areas of the plate.
	<ol>
		<li>Begin by placing the scalpel perpendicular (90&#176; angle) and 5 mm from the edge of the culture plate, and then roll the scalpel from this perpendicular angle to about an angle of 15&#176; (see Figure 1). 
		Note: This is achieved by pinching the scalpel between the index finger and thumb. Use gravity to allow the scalpel to gently go from the 90&#176; to 15&#176; position (as the rounded blade simply rolls over the cells). This motion will leave a visually noticeable indentation line.</li>
	</ol>
	</li>
	<li>Incubate the cells with the LY solution for 3 min at room temperature.</li>
	<li>Decant or aspirate the LY and rinse three times with 3 ml of PBS to remove all extracellular dye to eliminate extracellular background fluorescence.</li>
	<li>Add 0.5 ml of 10% phosphate buffered formalin solution to fix the cells. 
	Note: After fixing in formalin, air dry the cells and store in the dark for up to two years with minimum photobleaching of the LY dye. When required, rehydrate with 10% formalin solution to visualize the fluorescent dye front.</li>
	<li>View the fixed cells using an epifluorescence microscope equipped with a dichroic cube for excitation/emission peaks of 428/536 nm at a magnification of 200X. Align all plates so that the indentation line is parallel to the horizontal field of vision. 
	Note: A FITC dichroic cube works well.
	<ol>
		<li>Capture the image with a CCD camera and supporting software.
		<ol>
			<li>Open the supporting software for the CCD camera and use the settings for auto exposure. Use the &#34;capture&#34; button to digitally acquire the image. Save the image as .tif files using the &#34;Save&#34; button.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Adaptation of the SL-DT Assay to Liver Tissue
<ol>
	<li>Remove approximately a 2 x 2 cm slice from the left lobe of a liver from a 5 week old, male Fischer 344 rat using dissection scissors and place it on a plastic weigh plate covered with wet gauze12.</li>
	<li>Pipette 0.5 ml of a PBS solution containing 1 mg/ml each of lucifer yellow and rhodamine-dextran (RD) onto the surface of the liver slice in the plastic weigh plate. 
	Note: The RD is a dye that cannot traverse gap junction channels and will mark the cells that were loaded.</li>
	<li>Make three to five incisions approximately 1 cm long on the surface of the liver slice that has the dye solution in the weigh plate with a sharp blade and then add additional dye sufficient to fill the incisions.</li>
	<li>Incubate the tissue for 3 min at room temperature on the plastic weigh plate.</li>
	<li>Rinse three times with 5 ml of PBS.</li>
	<li>Fix the tissue overnight in 30 ml of 10% phosphate buffered formalin in a 50 ml conical centrifuge tube in the dark at room temperature.</li>
	<li>The following day, wash the slices with water, trim tissue around the incision with dissecting scissors into 1 x 1 x 0.5 cm3 strips and then use standard techniques to embed the slices in paraffin13.</li>
	<li>Section the slices perpendicular to the incision line to thickness of 5 &#181;m and store the samples in the dark until ready to be imaged using standard sectioning techniques with a microtome13.</li>
	<li>Use an epifluorescence microscope equipped with a CCD digital camera to visualize and capture the images of the fluorescent dye fronts according to section 1.107.</li>
</ol>
3. Quantifying GJIC
<ol>
	<li>Measure the fluorescent dye spread using a morphometric software package (e.g., ImageJ).
	<ol>
		<li>Click the &#34;File&#34; tab and then click &#34;open&#34; to open the saved image.</li>
		<li>Click on the &#34;Free Hand Tool&#34; in the Tool Bar Tab to trace the outline of the dye front.</li>
		<li>Click the &#34;Analyze&#34; tab and then click &#34;Measure&#34;. 
		Note: This will generate an area value in a spread sheet, which can be copied into other spread sheet programs if desired.</li>
	</ol>
	</li>
	<li>Using a spreadsheet program, compute the fraction of the control (FOC) by dividing the area of the dye spread in the experimental plate by the area the dye travelled in the control plate using the following equation: 
	<img alt="Equation1" src="/files/ftp_upload/54281/54281equation1.jpg" /> 
	Ae = area of the dye spread in cells exposed to an experimental variable, such as cells treated with a chemical at a specific dose or time 
	Ac = area of the dye spread in the control, which are cells treated with the vehicle used to dissolve the chemical of interest 
	Note: To determine the effect of the vehicle, Ae would be the area of the dye spread in cells treated by the vehicle and Ac would be the area of the dye spread in cells not treated by the vehicle. The effect of the vehicle should preferably be less than 10%.</li>
</ol>

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The authors have nothing to disclose.

The SL-DT assay is a simple and versatile technique in measuring GJIC, but there are several critical concerns that should be accounted for in designing appropriate experimental protocols. For robust measurements of GJIC using the SL-DT assay there must be a good dye spread of the LY through gap junctions of the cells. At minimum, an adequate time should be selected to assure that the dye spreads through eight or more rows of cells from the scalpel loaded cells in both directions. Also, for the ease of measurement of the...

The overall goal of this method is to provide a simple, comprehensive and relatively inexpensive technique to assess the potential toxicities of compounds. This is an in vitro approach that can be used in multiple cell lines. Standard cell biology labs equipped with epifluorescence microscopes can conduct research using this assay.
Our basic knowledge of cell functions has been highly dependent on in vitro bioassays, and has become an essential component in toxicological assessments of pharmaceuticals, environmental pollutants, and food born contaminants. Unfortunately, there is no single in vitro bioassay system that can comprehensively meet the demands for all toxicological assessments. Many in vitro assays are designed and optimized to evaluate as well as assess a specific biochemical or molecular endpoint. These are quite often combined in a high throughput set up to reflect a perturbation of a certain signal transduction pathway, such as estrogen-receptor signaling1. This strategy has been quite successful, but the extensive number of signal transduction pathways involved in gene expression makes the task of choosing a specific signaling pathway to assess quite complex. High through-put protocols are currently being developed and used to simultaneously measure numerous signaling pathways, which has been one approach to overcome some of the limitations of single assays. However, not all signaling pathways have been successfully incorporated into more comprehensive approaches, plus new signaling pathways are constantly being discovered that further complicates this assessment process. Using extensive numbers of in vitro approaches, particularly high through-put systems, for comprehensive toxicological assessments are also very expensive and are not conducive to most single investigator led research projects.
GJIC is a process tightly controlled by changes in voltage, calcium concentration, pH, redox balance, regulated by major intracellular signal transduction pathways and interactions with membrane and cytoskeleton proteins2,3. Thus, inhibition of GJIC can reflect different types of cellular stress, disruption of different cellular functions, or perturbations of different signal transduction pathways. Another approach to overcome the use of limited signal transduction bioassays is to take advantage of the biological phenomena that many, if not most, signal transduction pathways are further modulated by cooperative intercellular signaling systems through gap junction channels4-8. Although intercellular signaling systems are also numerous and under multiple pathway control, the intercellular signaling through gap junction channels is ultimately a function of the channels being opened, partially closed or completely closed. This provides an endpoint that can be easily measured using various in vitro bioassay systems7. Considering that the homeostatic set point of a tissue requires open channels, determining the effect of compounds on gap junctional intercellular communication (GJIC) is a more comprehensive approach in determining potential toxic effects of compounds4,8. In essence, this critical biological phenomenon that plays a central role in coordinating multiple signal transduction events controlling gene expression allows for a broad assessment of toxic effects. Thus, bioassays that assess GJIC are an excellent starting point to evaluate the toxic potential of compounds.
The most extensive techniques used to assess GJIC are based on preloading cells with a fluorescent probe and then monitoring the migration of the dye from the loaded cell or cells to adjacent cells. Techniques to preload the dye have involved microinjection9, scrape loading10, and methyl esters of the probes11. The scalpel loading-fluorescent dye transfer (SL-DT) method is a modification of the scrape load - dye transfer assay developed by El Fouly10. Rather than the more invasive scrape, the scalpel loading method of this report involves a gentle roll of a scalpel with a round blade through a monolayer of cells that minimize invasive damage (Figure 1). The advantages of this technique are the toxicological assessment of a population of cells rather than single cells of the microinjection assay. Furthermore, the simplicity of this assay allows for rapid detection of multiple plates in a short time whereas methods using microinjection techniques and techniques that use methyl esters of fluorescent probes are significantly more time consuming and require considerably higher skill level.
Although there is no single method to meet all the needs of studying GJIC; the SL-DT assay is a simple, fairly inexpensive and versatile assay that can meet many of the needs for initial assessments of toxicities of various compounds. Major advantages include: simplicity, no special need for equipment or skills that are required for other methods such as microinjection, fluorescent recovery after photobleaching (FRAP) assay and local activation of molecular fluorescent probe assays, a rapid and simultaneous assessment of GJIC in a large number of cells, conducive to a high throughput set-up with automated fluorescence microscopy imaging and analysis, as well as its adaptability for in vivo studies.

<table><tbody><tr><td>WB-F344 rat liver epithelial cells</td><td>From Drs. J. W. Grisham and M. S. Tsao of the University of North Carolina (Chapel Hill, NC)</td><td>none</td><td>Provided by Drs. J. W. Grisham and M. S. Tsao University of North Carolina-Chapel Hill-NC</td></tr><tr><td>35 mm Culture Plates</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; culture flasks</td><td>Corning</td><td>430639</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; culture flasks</td><td>Corning</td><td>430641</td><td></td></tr><tr><td>D-medium, an Eagles modified medium&amp;nbsp;</td><td>ThermoFisher/GIBCO&amp;nbsp;</td><td>Formula No. 78-5470EF</td><td></td></tr><tr><td>fetal bovine serum</td><td>ThermoFisher/GIBCO&amp;nbsp;</td><td>10437</td><td></td></tr><tr><td>0.25% trypsin-EDTA&amp;nbsp;</td><td>ThermoFisher/GIBCO&amp;nbsp;</td><td>15050</td><td></td></tr><tr><td>phosphate buffered saline</td><td>homemade</td><td>see below for ingredient cat#&#039;s</td><td>137 mM NaCl, 2.7 mM KCl, 10 mM Na&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 2 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>KCl</td><td>JT Baker - Mallinckrodt&amp;nbsp;</td><td>3040-01</td><td></td></tr><tr><td>NaCl</td><td>JT Baker - Mallinckrodt&amp;nbsp;</td><td>3624-05</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>JT Baker - Mallinckrodt&amp;nbsp;</td><td>3819--01</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>JT Baker - Mallinckrodt&amp;nbsp;</td><td>3246-01</td><td></td></tr><tr><td>Lucifer Yellow CH, lithium salt</td><td>Sigma-Aldrich Chemical</td><td>L0259</td><td></td></tr><tr><td>rhodamine-dextran</td><td>Sigma-Aldrich Chemical</td><td>R9379</td><td></td></tr><tr><td>1-methylanthracene</td><td>Sigma-Aldrich Chemical</td><td></td><td></td></tr><tr><td>phenanthrene</td><td>Sigma-Aldrich Chemical</td><td>P11409</td><td></td></tr><tr><td>resveratrol</td><td>Sigma-Aldrich Chemical</td><td>R5010</td><td></td></tr><tr><td>D609</td><td>Tocris Bioscience&amp;nbsp;</td><td>1437</td><td></td></tr><tr><td>acetonitril</td><td>EMD</td><td>AX0145-1</td><td></td></tr><tr><td>37% solution formaldehyde</td><td>JT Baker - Mallinckrodt&amp;nbsp;</td><td>2106-01</td><td></td></tr><tr><td>#20 surgical blade</td><td>Fine Science Tools Inc.&amp;nbsp;</td><td>10317-14</td><td></td></tr><tr><td>50 ml conical sterile tubes</td><td>Thermo scientific&amp;nbsp;</td><td>339652</td><td></td></tr><tr><td>Nikon epifluorescence microscope&amp;nbsp;</td><td>Nikon -Mager Scientific</td><td>Eclipse TE300</td><td></td></tr><tr><td>Nikon FITC dichroic cube</td><td>Nikon -Mager Scientific</td><td>96107</td><td></td></tr><tr><td>CCD camera</td><td>Nikon -Mager Scientific</td><td>Nikon Cool Snap EZ CCD</td><td></td></tr><tr><td>imaging system</td><td>Nikon -Mager Scientific</td><td>Nikon NIS-Elements F2.2 imaging system.</td><td></td></tr><tr><td>ImageJ</td><td>National Institute of Health</td><td>http://imagej.nih.gov/ij/</td><td></td></tr></tbody></table>

gap junctional intercellular communication: a functional biomarker to assess adverse effects of toxicants and toxins, and health benefits of natural products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

Interruption of GJIC has been extensively used as a biomarker for identifying toxic compounds at the nongenotoxic, epigenetic level of gene control that induces adverse health effects14. For example, polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants of the environment but vary in their epigenetic toxicities as a function of their molecular structures15. The lower molecular weight PAHs are typically found at relatively higher concentrations than the ...

Watch this Scientific Journal Video about Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products 

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

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14.9K Views.  Michigan State University.  Dit protocol beschrijft een scalpel loading-fluorescerende kleurstof overdracht techniek die intercellulaire communicatie meet door middel van gap junction kanalen. Spleetovergangen intercellulaire communicatie is een belangrijke cellulaire proces waardoor weefsel homeostase wordt onderhouden en verstoring van deze cell signaling heeft nadelige gevolgen voor de gezondheid. 

Video: Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

Developmental Biology

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood cell (RBC) as adhesion sensor and presenting cell for one of the interacting molecules. It employs micromanipulation to bring the RBC into contact with another cell that expresses the other interacting molecule with precisely controlled area and time to enable bond formation. The adhesion event is detected as RBC elongation upon pulling the two cells apart. By controlling the density of the ligands immobilized on the RBC surface, the probability of adhesion is kept in mid-range between 0 and 1. The adhesion probability is estimated from the frequency of adhesion events in a sequence of repeated contact cycles between the two cells for a given contact time. Varying the contact time generates a binding curve. Fitting a probabilistic model for receptor-ligand reaction kinetics1 to the binding curve returns the 2D affinity and off-rate.
The assay has been validated using interactions of Fc&gamma; receptors with IgG Fc1-6, selectins with glycoconjugate ligands6-9, integrins with ligands10-13, homotypical cadherin binding14, T cell receptor and coreceptor with peptide-major histocompatibility complexes15-19.
The method has been used to quantify regulations of 2D kinetics by biophysical factors, such as the membrane microtopology5, membrane anchor2, molecular orientation and length6, carrier stiffness9, curvature20, and impingement force20, as well as biochemical factors, such as modulators of the cytoskeleton and membrane microenvironment where the interacting molecules reside and the surface organization of these molecules15,17,19.
The method has also been used to study the concurrent binding of dual receptor-ligand species3,4, and trimolecular interactions19 using a modified model21.
The major advantage of the method is that it allows study of receptors in their native membrane environment. The results could be very different from those obtained using purified receptors17. It also allows study of the receptor-ligand interactions in a sub-second timescale with temporal resolution well beyond the typical biochemical methods.
To illustrate the micropipette adhesion frequency method, we show kinetics measurement of intercellular adhesion molecule 1 (ICAM-1) functionalized on RBCs binding to integrin &alpha;L&beta;2 on neutrophils with dimeric E-selectin in the solution to activate &alpha;L&beta;2.

Adhesion Frequency Assay for In Situ Kinetics Analysis of Cross-Junctional Molecular Interactions at the Cell-Cell Interface

An adhesion frequency assay for measuring receptor-ligand interaction kinetics when both molecules are anchored on the surfaces of the interacting cells is described. This mechanically-based assay is exemplified using a micropipette-pressurized human red blood cell as adhesion sensor and integrin &alpha;L&beta;2 and intercellular adhesion molecule-1 as interacting receptors and ligands.

The micropipette adhesion assay was developed in 1998 to measure two-dimensional (2D) receptor-ligand binding kinetics1. The assay uses a human red blood ...

Bioengineering

Human In-Vivo Bioassay for the Tissue-Specific Measurement of Nociceptive and Inflammatory Mediators

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via aseptically inserted microdialysis catheters (Dermal Dialysis, Erlangen, Germany). Illustrated in this example is the collection of interstitial fluid from experimentally inflamed skin in human volunteers. Sample collection can be combined with other experimental tests. For example, the simultaneous assessment of locally released biochemicals and subjective sensitivity to painful stimuli in experimentally inflamed skin provides the critical biochemical-behavioral link to identify biomarkers of pain and inflammation. Presented assay in the living human organism allows for mechanistic insight into tissue-specific processes underlying pain and/or inflammation. The method is also well suited to examine the effectiveness of existing or novel interventions - such as new drug candidates - targeting the treatment of painful and/or inflammatory conditions.
This article will provide a detailed description on the use of microdialysis techniques for collecting interstitial fluid from experimentally inflamed skin lesion of human study subjects. Interstitial fluid samples are typically processed with aid of multiplex bead array immunoassays allowing assaying up to 100 analytes in samples as small in volume as 50 microliters.


A technique is presented for the in-vivo collection of interstitial fluid samples from pertinent tissue sites (here, experimentally inflamed skin) for the measurement of biochemicals mediating pain and inflammation.

This in-vivo human bioassay can be used to study human volunteers and patients. Samples are collected from pertinent tissue sites such as the skin via ...

Real-Time Impedance-based Cell Analyzer as a Tool to Delineate Molecular Pathways Involved in Neurotoxicity and Neuroprotection in a Neuronal Cell Line

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic effects of drugs and downstream pathways involved would help gain insight into the molecular mechanisms of neuroprotection/neurotoxicity and could potentially facilitate drug development. However, many existing in vitro toxicity assays have major limitations &#8211; most assess neurotoxicity and neuroprotection at a single time point, not allowing to observe the time-course and kinetics of the effect. Furthermore, the opportunity to collect information about downstream signaling pathways involved in neuroprotection in real-time would be of great importance. In the current protocol we describe the use of a real-time impedance-based cell analyzer to determine neuroprotective effects of serotonin 2A (5-HT2A) receptor agonists in a neuronal cell line under label-free and real-time conditions using impedance measurements. Furthermore, we demonstrate that inhibitors of second messenger pathways can be used to delineate downstream molecules involved in the neuroprotective effect. We also describe the utility of this technique to determine whether an effect on cell proliferation contributes to an observed neuroprotective effect. The system utilizes special microelectronic plates referred to as E-Plates which contain alternating gold microelectrode arrays on the bottom surface of the wells, serving as cell sensors. The impedance readout is modified by the number of adherent cells, cell viability, morphology, and adhesion. A dimensionless parameter called Cell Index is derived from the electrical impedance measurements and is used to represent the cell status. Overall, the real-time impedance-based cell analyzer allows for real-time, label-free assessment of neuroprotection and neurotoxicity, and the evaluation of second messenger pathways involvement, contributing to more detailed and high-throughput assessment of potential neuroprotective compounds in vitro, for selecting therapeutic candidates.

Improved in vitro neurotoxicity assays would aid the identification of new neuroprotective compounds. The utility of a real-time impedance-based cell analyzer to determine cytotoxicity and cytoprotection in neuronal cell lines and to delineate the involvement of second messenger pathways, thus gaining insight in the mechanism of neuroprotection is presented.

Many brain-related disorders have neuronal cell death involved in their pathophysiology. Improved in vitro models to study neuroprotective or neurotoxic ...

Neuroscience

Evaluating MOF-Induced Cell Behavior Changes by Electric Cell-Substrate Sensing

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of MOFs in biomedical and industrial applications has led to concerns regarding their safety. Herein, the profile of a selected MOF, a zeolitic imidazole framework, was evaluated upon exposure to human lung epithelial cells. The platform for evaluation was a real-time technique (i.e., electric cell-substrate impedance sensing [ECIS]). This study identifies and discusses some of the deleterious effects of the selected MOF on the exposed cells. Furthermore, this study demonstrates the benefits of using the real-time method versus other biochemical assays for comprehensive cell evaluations. The study concludes that observed changes in cell behavior could hint at possible toxicity induced upon exposure&#160;to MOFs of different physicochemical characteristics and the dosage of those frameworks being used. By understanding changes in cell behavior, one foresees the ability to improve safe-by-design strategies of MOFs to be used for biomedical applications by specifically tailoring their physicochemical characteristics.

Author Spotlight: Advances in Evaluating Human Lung Epithelial Cells' Response to Metal-Organic Frameworks 

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Accessing the Cytotoxicity and Cell Response to Biomaterials

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Gr&#252;nwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...

Medicine

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

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