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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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. 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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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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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>
</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. 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.

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

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media.
Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods13. Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo.
In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods.
We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.

Unfixed frozen tissue samples embedded in Optimal Cutting Temperature medium (OCT) can be used to study natural distribution and glycosylation of secreted mucus. In this approach tissue processing is minimal and the natural presentation of glycolipids, mucins and glycan-epitopes is preserved. Tissue sections can be analyzed by immunohistochemistry using fluorescence or chromogenic detection.

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by ...

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. ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

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 ...

At-Risk Butterfly Captive Propagation Programs to Enhance Life History Knowledge and Effective Ex Situ Conservation Techniques

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. Research on such captive populations can also yield valuable data to address key information gaps about the behavior, life history, and ecology of the target taxa. We describe a protocol for captive propagation of the federally endangered Cyclargus thomasi bethunebakeri that can be used as a model for other at-risk butterfly ex situ programs, especially those in the family Lycaenidae. We further provide a simple and straightforward protocol for recording various life history metrics that can be useful for informing ex situ methodologies as well as adapted for laboratory studies of other lepidoptera.

Here, we present protocols for 1) the laboratory captive propagation of the federally endangered Miami blue butterfly (Cyclargus thomasi bethunebakeri), and 2) assessing basic life history information such as immature development time and number of larval stadia. Both methods can be adapted for use with other ex situ conservation programs.

Improving knowledge of ex situ best practices for at-risk butterflies is important for generating successful conservation and recovery program outcomes. ...

Field Identification of Matricaria chamomilla using a Portable qPCR System

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Field Identification of Matricaria chamomilla using a Portable qPCR System

Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses.

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...