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...

<ol>
	<li>Rotroff, D. 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=Predictive+endocrine+testing+in+the+21st+century+using+in+vitro+assays+of+estrogen+receptor+signaling+responses.">Predictive endocrine testing in the 21st century using in vitro assays of estrogen receptor signaling responses.</a> Environ.Sci.Technol. 48 (15), 8706-8716 (2014).</li><li>Axelsen, L. N., Calloe, K., Holstein-Rathlou, N. H., Nielsen, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+the+complexity+of+communication:+regulation+of+gap+junctions+by+post-translational+modification.">Managing the complexity of communication: regulation of gap junctions by post-translational modification.</a> Front Pharmacol. 4, 130 (2013).</li><li>Nielsen, M. S., 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=Gap+junctions.">Gap junctions.</a> Compr.Physiol. 2 (3), 1981-2035 (2012).</li><li>Sovadinova, I., 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=Phosphatidylcholine+specific+PLC-induced+dysregulation+of+gap+junctions,+a+robust+cellular+response+to+environmental+toxicants,+and+prevention+by+resveratrol+in+a+rat+liver+cell+model.">Phosphatidylcholine specific PLC-induced dysregulation of gap junctions, a robust cellular response to environmental toxicants, and prevention by resveratrol in a rat liver cell model.</a> PLoS.One. 10 (5), e0124454 (2015).</li><li>Trosko, J. E., Chang, C. C., Travis, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biologically Based Methods for Cancer Risk Assessment. , 165-179 (1989).</li><li>Trosko, J. E., Mendelsohn, M. D., Peeters, J. P., Normandy, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomarkers and occupational health: progess and perspectives. , 264-274 (1995).</li><li>Upham, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+integrative+signaling+through+gap+junctions+in+toxicology.">Role of integrative signaling through gap junctions in toxicology.</a> Curr.Protoc.Toxicol. 47, 2.18:2.18.1-2.18:2.18.18 (2011).</li><li>Vinken, 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=Connexins+and+their+channels+in+cell+growth+and+cell+death.">Connexins and their channels in cell growth and cell death.</a> Cell.Signal. 18 (5), 592-600 (2006).</li><li>Browne, C. L., Wiley, H. S., Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-follicle+cell+gap+junctions+in+Xenopus+laevis+and+the+effects+of+gonadotropin+on+their+permeability.">Oocyte-follicle cell gap junctions in Xenopus laevis and the effects of gonadotropin on their permeability.</a> Science. 203 (4376), 182-183 (1979).</li><li>El-Fouly, M. H., Trosko, J. E., Chang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrape-loading+and+dye+transfer.+A+rapid+and+simple+technique+to+study+gap+junctional+intercellular+communication.">Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication.</a> Exp.Cell Res. 168 (2), 422-430 (1987).</li><li>Wade, M. H., Trosko, J. E., Schindler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence+photobleaching+assay+of+gap+junction-mediated+communication+between+human+cells.">A fluorescence photobleaching assay of gap junction-mediated communication between human cells.</a> Science. 232 (4749), 525-528 (1986).</li><li>Upham, B. L., 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=Structure-activity-dependent+regulation+of+cell+communication+by+perfluorinated+fatty+acids+using+in+vivo+and+in+vitro+model+systems.">Structure-activity-dependent regulation of cell communication by perfluorinated fatty acids using in vivo and in vitro model systems.</a> Environ. Health Perspect. 117 (4), 545-551 (2009).</li><li>Trosko, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctional+intercellular+communication+as+a+biological+&amp;#34;Rosetta+stone&amp;#34;+in+understanding,+in+a+systems+biological+manner,+stem+cell+behavior,+mechanisms+of+epigenetic+toxicology,+chemoprevention+and+chemotherapy.">Gap junctional intercellular communication as a biological &amp;#34;Rosetta stone&amp;#34; in understanding, in a systems biological manner, stem cell behavior, mechanisms of epigenetic toxicology, chemoprevention and chemotherapy.</a> J. Membr. Biol. 218 (1-3), 93-100 (2007).</li><li>Upham, B. L., Weis, L. M., Trosko, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulated+gap+junctional+intercellular+communication+as+a+biomarker+of+PAH+epigenetic+toxicity:+structure-function+relationship.">Modulated gap junctional intercellular communication as a biomarker of PAH epigenetic toxicity: structure-function relationship.</a> Environ.Health Perspect. 106, 975-981 (1998).</li><li>Upham, B. L., 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=Tumor+promoting+properties+of+a+cigarette+smoke+prevalent+polycyclic+aromatic+hydrocarbon+as+indicated+by+the+inhibition+of+gap+junctional+intercellular+communication+via+phosphatidylcholine-specific+phospholipase.">Tumor promoting properties of a cigarette smoke prevalent polycyclic aromatic hydrocarbon as indicated by the inhibition of gap junctional intercellular communication via phosphatidylcholine-specific phospholipase.</a> C. Cancer Sci. 99 (4), 696-705 (2008).</li><li>Sai, K., Kanno, J., Hasegawa, R., Trosko, J. 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Cancer. 78 (4), 491-495 (1998).</li><li>Trosko, J. E., Tai, M. H., Dittmar, T., Zaenkar, K. S., Schmidt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+and+inflammation:+Impacts+on+oncogenesis.+Impacts+on+Oncogenesis.">Infections and inflammation: Impacts on oncogenesis. Impacts on Oncogenesis.</a> Contrib Microbiol. , 45-65 (2006).</li><li>Trosko, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+stem+cells+as+targets+for+the+aging+and+diseases+of+aging+processes.">Human stem cells as targets for the aging and diseases of aging processes.</a> Med. Hypotheses. 60 (3), 439-447 (2003).</li><li>JE, T. r. o. s. k. o., Dittmar, T., Zaenkar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stem Cells and Cancer. , 147-188 (2008).</li><li>Osgood, R. S., 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=Polycyclic+aromatic+hydrocarbon-induced+signaling+events+relevant+to+inflammation+and+tumorigenesis+in+lung+cells+are+dependent+on+molecular+structure.">Polycyclic aromatic hydrocarbon-induced signaling events relevant to inflammation and tumorigenesis in lung cells are dependent on molecular structure.</a> PLoS. One. 8 (6), e65150 (2014).</li><li>Weis, L. M., Rummel, A. M., Masten, S. J., Trosko, J. E., Upham, B. 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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 border="0" cellpadding="0" cellspacing="0" width="1238">
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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:312px;">WB-F344 rat liver epithelial cells</td>
			<td style="width:217px;">From Drs. J. W. Grisham and M. S. Tsao of the University of North Carolina (Chapel Hill, NC)</td>
			<td style="width:279px;">none</td>
			<td style="width:431px;">Provided by Drs. J. W. Grisham and M. S. Tsao University of North Carolina-Chapel Hill-NC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">35 mm Culture Plates</td>
			<td style="width:217px;">Corning</td>
			<td style="width:279px;">430165</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">25 cm2 culture flasks</td>
			<td style="width:217px;">Corning</td>
			<td style="width:279px;">430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">75 cm2 culture flasks</td>
			<td style="width:217px;">Corning</td>
			<td style="width:279px;">430641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-medium, an Eagles modified medium&#160;</td>
			<td style="width:217px;">ThermoFisher/GIBCO&#160;</td>
			<td style="width:279px;">Formula No. 78-5470EF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">fetal bovine serum</td>
			<td style="width:217px;">ThermoFisher/GIBCO&#160;</td>
			<td style="width:279px;">10437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% trypsin-EDTA&#160;</td>
			<td style="width:217px;">ThermoFisher/GIBCO&#160;</td>
			<td style="width:279px;">15050</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:312px;">phosphate buffered saline</td>
			<td style="width:217px;">homemade</td>
			<td style="width:279px;">see below for ingredient cat#&#39;s</td>
			<td>137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 2 mM KH2PO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">KCl</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3040-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">NaCl</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3624-05</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Na2HPO4</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3819--01</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td style="width:217px;">JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">3246-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Lucifer Yellow CH, lithium salt</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">L0259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">rhodamine-dextran</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">R9379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">1-methylanthracene</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">phenanthrene</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">P11409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">resveratrol</td>
			<td style="width:217px;">Sigma-Aldrich Chemical</td>
			<td style="width:279px;">R5010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">D609</td>
			<td>Tocris Bioscience&#160;</td>
			<td style="width:279px;">1437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">acetonitril</td>
			<td>EMD</td>
			<td style="width:279px;">AX0145-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">37% solution formaldehyde</td>
			<td>JT Baker - Mallinckrodt&#160;</td>
			<td style="width:279px;">2106-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">#20 surgical blade</td>
			<td>Fine Science Tools Inc.&#160;</td>
			<td>10317-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">50 mL conical sterile tubes</td>
			<td>Thermo scientific&#160;</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon epifluorescence microscope&#160;</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td style="width:279px;">Eclipse TE300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon FITC dichroic cube</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td style="width:279px;">96107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;CCD camera&#160;</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td style="width:279px;">Nikon Cool Snap EZ CCD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;imaging system.</td>
			<td style="width:217px;">Nikon -Mager Scientific</td>
			<td>Nikon NIS-Elements F2.2 imaging system.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:312px;">Image J</td>
			<td style="width:217px;">National Institute of Health</td>
			<td style="width:279px;">http://imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
	</tbody>
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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 ...

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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 ...

The overall goal is to provide a simple assay to measure gap junctional intercellular communication through a population of mammalian cells that can serve a broad range of in vitro pharmacological and toxicological needs. This method can help answer key questions in the toxicology field, such as whether a compound affects the development or maintenance of a tissue, by disregulating gap junctional intercellular communication. The main advantage of this technique is that it offers a simple and quick assessment of gap junction function in a large population of cells in response to toxicants and toxins.Demonstrating the procedure will be Ajna Uzuni, an undergraduate research assistant from my laboratory. To begin the experiment, seed three times 10 to the fifth WB-F344 rat liver epithelial cells onto 35 millimeter diameter culture plates containing Eagle's modified medium plus 5%fetal-bovine serum. Culture the cells in an incubator until they reach 100%confluence and continue with the experimental treatment of the cells.To continue with dose-response experiments, add 10 microliters and 20 microliters of a previously prepared, two millimolar stock solution of phenanthrene in 100%acetonitrile, and four microliters, six microliters, and eight microliters of a 20 millimolar stock solution of phenanthrene in 100%acetonitrile to three plates of cells containing two milliliters of growth medium for each added volume of stock solution. Next, add four microliters, six microliters, eight microliters, 10 microliters, and 20 microliters of a 100%solution of acetonitrile to three plates of cells containing two milliliters of growth medium for each added volume of acetonitrile solution to serve as the vehicle control. After adding the acetonitrile, incubate the plates.To perform time response experiments, add seven microliters of a 20 millimolar stock solution of phenanthrene in 100%acetonitrile to three plates of cells, containing two milliliters of growth medium for each time point. Then, add seven microliters of 100%acetonitrile to three plates of cells, containing two milliliters of growth medium for each time point, to serve as the vehicle control. Incubate the plates for each designated time point.Discard the culture medium by gently pouring off the medium. Rinse the cells with three milliliters of PBS, and either decant or aspirate between rinses. Pipette one milliliter of one milligram per milliliter lucifer yellow dye dissolved in PBS into each cell plate.Preload the dye into the cells by pinching the scalpel between the index finger and thumb. Place the scalpel perpendicular to and five millimeters from the edge of the culture plate and then use gravity to allow the scalpel to gently roll from this perpendicular angle to an angle of 15 degrees. Roll the blade through a population of cells in three different areas of the total plate.Next, incubate the cells with the LY solution for three minutes at room temperature. Decant or aspirate the LY and rinse the cells to remove all extracellular dye and eliminate extra cellular background fluorescence. Then, add 0.5 milliliters of a 4%phosphate buffered formalin solution to fix the cells.View the fixed cells using an epifluorescence microscope at 200x magnification. Align all of the plates so that the indentation line is parallel to the horizontal field of vision. A series of SL-DT images were generated of WB-F344 rat liver epithelial cells treated with increasing doses of phenanthrene.The decrease in lucifer yellow fluorescent dye migration is caused by increasing doses of phenanthrene, demonstrating the increasing toxicant-induced inhibition of gap junctional intercellular communication, or GJIC. The fraction of the control, or FOC values, obtained from the SL-DT images were averaged, statistically analysed, and then graphed. Following this procedure, other methods like western blots of single transduction proteins can be performed in order to answer additional questions.Like, are these compounds activating mitogenic events of cell perforation?

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14.5K Görüntüleme.  Michigan State University. The overall goal is to provide a simple assay to measure gap junctional intercellular communication through a population of mammalian cells that can serve a broad range of in vitro pharmacological and toxicological needs. This method can help answer key questions in the toxicology field, such as whether a compound affects the development or maintenance of a tissue, by disregulating gap junctional intercellular communication. The main advantage of this technique is that it offers a simple and ...

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