Name: salinity-dependent toxicity assay of silver nanocolloids using medaka eggs
Uploaded: 2016-03-18T13:00:00
Description: Watch this Scientific Journal Video about Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs at JoVE.com

We are grateful to Ms. Kaori Shimizu and Mr. Masaki Takasu of the Graduate School of Life Sciences, Toyo University, for their technical support. This project was supported by research grants from the Special Research Foundation and Bio-Nano Electronics Research Centre of Toyo University (to SK); by the Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan (to SK); by the New Project Fund for Risk Assessments, from the Ministry of Economy, Trade and Industry (to SK); by a Grant-in-Aid for Challenging Exploratory Research (award 23651028 to SK); by a Grant-in-Aid for Scientific Research (B) and (C) (award 23310026 and 26340030 to SK); and by a Grant-in-Aid for Strategic Research Base Project for Private Universities (award S1411016 to SK) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

The Japanese medaka used in this study were treated humanely in accordance with the institutional guidelines of Toyo University, with due consideration for the alleviation of distress and discomfort.
1. Silver Nanocolloids (SNCs)
<ol>
	<li>Purchase purified SNCs (20 mg/L&#8722;1, 99.99% purity, particle mean diameter about 28.4 &#177; 8.5 nm suspended in distilled water).</li>
	<li>Validate the purity and concentration of the silver by inductively coupled plasma mass spectrometry ...

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> OECD Guidelines for the Testing of Chemicals, Section 2 Effects on Biotic Systems. , (2015).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> National Coastal Condition Report. , (2001).</li><li>Sakaizumi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+inorganic+salts+on+mercury-compound+toxicity+to+the+embryos+of+the+Medaka,+Oryzias+latipes.">Effect of inorganic salts on mercury-compound toxicity to the embryos of the Medaka, Oryzias latipes.</a> J. Fac. Sci. Univ. Tokyo. 14 (4), 369-384 (1980).</li><li>Sumitani, K., Kashiwada, S., Osaki, K., Yamada, M., Mohri, S., Yasumasu, 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=Medaka+(Oryzias+latipes)+Embryo+toxicity+of+treated+leachate+from+waste-landfill+sites.">Medaka (Oryzias latipes) Embryo toxicity of treated leachate from waste-landfill sites.</a> J. Jpn. Soc. Waste Manage. Exp. 15 (6), 472-479 (2004).</li><li>Kashiwada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+Nanoparticles+in+the+See-through+Medaka+(Oryzias+latipes).">Distribution of Nanoparticles in the See-through Medaka (Oryzias latipes).</a> EHP. 114 (11), 1697-1702 (2006).</li><li>Iwamatsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Integrated Book for the Biology of the Medaka. , (2006).</li><li>Miyamoto, T., Machida, T., Kawashima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+environmental+salinity+on+the+development+of+chloride+cells+of+freshwater+and+brackish-water+medaka,+Oryzias+latipes.">Influence of environmental salinity on the development of chloride cells of freshwater and brackish-water medaka, Oryzias latipes.</a> Zoo. Sci. 3 (5), 859-865 (1986).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> XSERIES 2 ICP-MS Getting Started Guide Revision B - 121 9590. , (2007).</li><li>Kashiwada, S., Ariza, M. E., Kawaguchi, T., Nakagame, Y., Jayasinghe, B. S., Gartner, K., 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=Silver+nanocolloids+disrupt+medaka+embryogenesis+through+vital+gene+expressions.">Silver nanocolloids disrupt medaka embryogenesis through vital gene expressions.</a> ES &amp; T. 46 (11), 6278-6287 (2012).</li><li>Iwamatsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+normal+development+in+the+medaka+Oryzias+latipes.">Stages of normal development in the medaka Oryzias latipes.</a> Mech. Dev. 121, 605-618 (2004).</li><li>Kataoka, C., Ariyoshi, T., Kawaguchi, H., Nagasaka, S., Kashiwada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salinity+increases+the+toxicity+of+silver+nanocolloids+to+Japanese+medaka+embryos.">Salinity increases the toxicity of silver nanocolloids to Japanese medaka embryos.</a> Environ. Sci.: Nano. 2, 94-103 (2014).</li><li>Shannon, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+effective+ionic+radii+and+systematic+studies+of+interatomic+distances+in+halides+and+chalcogenides.">Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides.</a> Acta Cryst. 32, 751-767 (1976).</li><li>Wakamatsu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Medaka Book, 6.1: Preparation of hatching enzyme. , (2015).</li></ol>

Medaka is a freshwater fish that is highly tolerant to seawater; it is not well known that the original natural habitat of this fish was saltwater off the Japanese coast6. Hence, medaka fish have well-developed chloride cells7. This unique property provides scientists with a new way to test the toxicity of chemicals in the environment as a function of salinity (freshwater to seawater) by using only a single species of fish.
To obtain medaka eggs at stage 21, eggs must be ...

Since the establishment of the Organisation for Economic Co-operation and Development (OECD) test guidelines for testing chemicals in 1979, 38 test guidelines have been published in Section 2 of the guidelines, Effects on Biotic Systems1. All of the aquatic organisms tested have been from freshwater habitats, namely freshwater plants; algae; invertebrates such as daphnia and chironomids; and fishes such as medaka, zebrafish, and rainbow trout. Compared to saltwater environments, freshwater environments are more directly affected by human economic and industrial activities. Therefore, freshwater environments have been prioritized for testing because they are at higher risk from pollution.
In coastal areas, including estuaries, salinities vary between brackish water and seawater conditions, and these areas are often polluted by industrial activity2. Coastal areas and their associated wetlands are characterized by high ecological biodiversity and productivity. Coastal ecosystems must therefore be protected from chemical pollution. However, there has been limited ecotoxicological research in brackish water and seawater habitats.
Sakaizumi3 studied the toxic interactions between methyl mercury and salinity in Japanese medaka eggs and found that increasing the osmotic pressure of the test solution enhanced the toxicity of the methyl mercury. Sumitani et al.4 used medaka eggs to investigate the toxicity of landfill leachate; they found that the osmotic equivalency of leachate to the eggs was the key to inducing abnormalities during embryogenesis. In addition, Kashiwada5 reported that plastic nanoparticles (39.4 nm in diameter) easily permeated through the medaka egg chorion under brackish conditions (15x embryo rearing medium (ERM)).
A typical small fish model, the Japanese medaka (Oryzias latipes) has been used in basic biology and ecotoxicology6. Japanese medaka can live in conditions ranging from freshwater to seawater because of their highly developed chloride cells7. They are therefore likely to be useful for testing in conditions with a wide range of salinities.

<table border="0" cellpadding="0" cellspacing="0" width="785">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Silver nanocolloids</td>
			<td style="width:188px;">Utopia Silver Supplements</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">NaCl</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31319-45</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">KCl</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">28513-85</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2&#183;2H2O</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">06730-15</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#183;7H2O</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">21002-85</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3&#160;</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31212-25</td>
			<td>For making ERM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AgNO3</td>
			<td>Nacalai Tesque, Inc.</td>
			<td style="width:119px;">31018-72</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter</td>
			<td>HORIBA, Ltd.</td>
			<td style="width:119px;">F-51S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Balance</td>
			<td>Mettler-Toledo International Inc.</td>
			<td style="width:119px;">MS204S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">medaka (Oryzias latipes) orange-red strain&#160;</td>
			<td colspan="2">National Institute for Environmental Studies</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">medaka flow-through culturing system</td>
			<td>Meito Suien Co.</td>
			<td>MEITOsystem</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Artemia salina nauplii eggs</td>
			<td>Japan pet design Co. Ltd</td>
			<td>4975677033759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aeration pomp</td>
			<td>Japan pet design Co. Ltd</td>
			<td>non-noise w300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Otohime larval &#946;-1</td>
			<td style="width:188px;">Marubeni Nissin Feed Co. Ltd</td>
			<td style="width:119px;">Otohime larval &#946;-1</td>
			<td>Artificial dry fish diet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting microscope</td>
			<td style="width:188px;">Leica microsystems</td>
			<td style="width:119px;">M165FC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micrometer</td>
			<td>Fujikogaku, Ltd.</td>
			<td>10450023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">incubator</td>
			<td>Nksystem</td>
			<td>TG-180-5LB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">shaker</td>
			<td style="width:188px;">ELMI Ltd.</td>
			<td style="width:119px;">Aizkraukles 21-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plastic plates</td>
			<td>Greiner CELLSTAR</td>
			<td style="width:119px;">M8562-100EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aluminum foil</td>
			<td>AS ONE Co.</td>
			<td style="width:119px;">6-713-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">stopwatch</td>
			<td>DRETEC Co. Ltd.</td>
			<td style="width:119px;">SW-111YE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-kDa membrane filter</td>
			<td>EMD Millipore Corporation</td>
			<td colspan="2">0.5-mL centrifugal-type filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50-mL Teflon beaker</td>
			<td>AS ONE Co.</td>
			<td style="width:119px;">33431097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Custom claritas standard</td>
			<td style="width:188px;">SPEXertificate</td>
			<td style="width:119px;">ZSTC-538</td>
			<td>For internal standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Custom claritas standard</td>
			<td style="width:188px;">SPEXertificate</td>
			<td style="width:119px;">ZSTC-622</td>
			<td>For external standard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">ultrapure nitric acid</td>
			<td style="width:188px;">Kanto Chemical Co.</td>
			<td style="width:119px;">28163-5B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">hydrogen peroxide&#160;</td>
			<td style="width:188px;">Kanto Chemical Co.</td>
			<td style="width:119px;">18084-1B</td>
			<td>for atomic absorption spectrometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">ICP-MS</td>
			<td>Thermo Scientific</td>
			<td colspan="2">Thermo Scientific X Series 2&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hot plate</td>
			<td>Tiger Co.</td>
			<td>CRC-A300</td>
			<td></td>
		</tr>
	</tbody>
</table>

salinity-dependent toxicity assay of silver nanocolloids using medaka eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

The effect of salinity on SNC toxicity was very obvious: the induction of deformity or death was salinity dependent (Figures 1 and 2). We measured phenotypic biomarkers (heart rate, eye size, full body length, and hatching rate) in SNC (10 mg/L&#8722;1)-exposed embryos. These phenotypic biomarkers revealed salinity-dependent SNC toxicity.
Heart rates ranged from 29.6 to 32.2 beats/15 ...

Watch this Scientific Journal Video about Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs at JoVE.com

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

The overall goal of this experiment is to test the salinity dependent toxicity of chemicals to aquatic organisms. This method can help answer key questions in the Ecotoxicology field, such as assessment of toxicity on the fresh water, brackish water, and sea water conditions. The main advantage of this technique, is that medaka are never asked to test toxicity of chemicals in different salinities.Additionally, the implication of this technique extend to other therapy or diagnosis of chemicals toxicity. Preparation of commerical silver nanocolloids in solution, at various salinities, is covered in the text protocol. As is medaka culturing and egg collection.Begin with rinsing six collections of Stage 21 medaka eggs, three times in test solution. Do this using ERMs, at a different concentration for each set of eggs. Now, from each rinsed collection, transfer 15 eggs to a different well of test solution, in a six-well plastic plate.Set this experiment up in triplicate, washing every group separately. Wrap the plates in foil, and incubate them at 25 degrees Celcius, in the dark. Until the eggs hatch, or for 14 days, whichever comes sooner.During the incubation, observe the eggs daily for any biological changes. And for any dead eggs. Change the test solution daily, along with making the observations.On the sixth day of observation, count the heart rate of each embryo over 15 seconds. Also on the sixth day, measure the diameter of each embryo's eyes, in each experimental condition, using a micrometer. Whenever the embryos hatch into larvae, measure their full body lengths, using a micrometer.Also, over the 14 days, keep count of the total eggs that hatch. To measure silver bioaccumulation, on the sixth day, collect the eggs'chorions, using the standard method. The dechorionated eggs can be analyzed, just as the SNC solution is analyzed, as described in the following section.To isolate the soluble silver from each SNC solution. first, filter the mixture of colloids and soluble silver out, using a three kilodalton membrane. Then, spin down the loaded filter at 14, 000 Gs, for 10 minutes, in a refrigerated centrifuge.From 50 microliters of the filtrate, measure the silver concentration by ICP-MS analysis. Load the aliquot into a 50 millileter Teflon beaker, and add two milliliters of ultrapure nitric acid to the beaker. Then, heat the mixture on a hot plate, at 110 degrees Celcius.Over about three hours of heating, let the solution nearly dry out. Once the beaker has cooled, completely dissolve the organic solids. Add two millilters of ultrapure nitric acid, and 0.5 milliliters of hydrogen peroxide, to the beaker.Then, heat the mixture again, until it has nearly dried out again. Now, dissolve the remaining residue in four milliliters of one percent ultrapure nitric acid, made in ultrapure water. Transfer the solution to a 15 milliliter centrifuge tube.Repeat the process of dissolving the remaining solids in the Teflon beaker, two more times, cooling the three collections into a single tube. Now, measure the silver concentration in the sample, using ICP-MS. Use the manufacturer's recommended protocol to analyze the samples.Be sure to use internal and external standards. The effect of salinity on SNC toxicity was easily apparent. The induction of deformity or death was clearly salinity-dependent.Various physiological measurements were made. Heart rates were unaffected until the salinity reached 30 X ERM. Body length was significantly shorter at salinities of 15 and 30 X ERM.Eye diameter dropped as well, consistent with body length. Hatching was less likely at 15 X ERM, and only occurred for two percent of eggs in 30 X ERM. Under the experimental solutions, water soluble metal complexes formed more readily as the salt concentration increased.Examination of the metal complexes in the embryos after six days showed that they increased in concentration in the embryos as they increased in concentration in the solution. While attempting this procedure, it's important to remember to adjust the pH of the test conditions, because silver ion release is facilitated by acidic condition.

salinity-dependent-toxicity-assay-silver-nanocolloids-using-medaka

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Developmental Biology

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 ...

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing cardiovascular progenitors during development and disease. While a number of factors have been implicated in epicardium-derived cell (EPDC) mobilization, the mechanisms governing their subsequent migration and differentiation are poorly understood. Here, we present in vitro and ex vivo strategies to study EPDC motility and differentiation. First, we describe a method of obtaining primary epicardial cells by outgrowth culture from the embryonic mouse heart. We also introduce a detailed protocol to assess three-dimensional migration of labeled EPDC in an organ culture system. We provide evidence using these techniques that genetic deletion of myocardin-related transcription factors in the epicardium attenuates EPDC migration. This approach serves as a platform to evaluate candidate modifiers of EPDC biology and could be used to develop genetic or chemical screens to identify novel regulators of EPDC mobilization that might be useful for cardiac repair.

Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

A single layer of epicardial cells lines the heart, providing paracrine factors that stimulate cardiomyocyte proliferation and directly contributing ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

HUVEC Tube-formation Assay to Evaluate the Impact of Natural Products on Angiogenesis

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the formation of new blood vessels and involve several signal transduction pathways. Here we show that the tube formation assay is a simple in vitro method to evaluate the impact of natural products on angiogenesis and to investigate the molecular mechanisms involved. In particular, in the presence of the water extract of Ruta graveolens (RGWE), endothelial cells are no longer able to form a cell-cell network and that the RGWE effects on human umbilical vein endothelial cell (HUVEC) tube formation is abolished by the constitutive activation of MEK.

Here, we evaluate the effects of the water extract of Ruta graveolens on vessel network formation by using a tube formation assay on a gelled basement matrix.

Angiogenesis is a phenomenon that includes different processes, such as endothelial cell proliferation, differentiation, and migration, that lead to the ...

Injecting Gryllus bimaculatus Eggs

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been developed in traditional model systems, it is difficult to manipulate genes or messenger RNA (mRNA) in most other organisms. At the same time, evolutionary and comparative approaches rely on an exploration of gene function in many different species, necessitating the development and adaptation of techniques for manipulating expression outside currently genetically tractable species. This protocol describes a method for injecting reagents into cricket eggs to assay the effects of a given manipulation on embryonic or larval development. Instructions for how to collect and inject eggs with beveled needles are described. This relatively straightforward technique is flexible and potentially adaptable to other insects. One can gather and inject dozens of eggs in a single experiment, and survival rates for buffer-only injections improve with practice and can be as high as 80%. This technique will support several types of experimental approaches including injection of pharmacological agents, in vitro capped mRNA to express genes of interest, double-stranded RNA (dsRNA) to achieve RNA interference, use of clustered regularly interspaced short palindromic repeats (CRISPR) in concert with CRISPR-associated protein 9 (Cas9) reagents for genomic modification, and transposable elements to generate transient or stable transgenic lines.

Injecting Gryllus bimaculatus Eggs

Here we present a protocol to inject cricket eggs, a technique which serves as a foundational method in many experiments in the cricket, including, but not limited to, RNA interference and genomic manipulation.

Altering gene function in a developing organism is central to different kinds of experiments. While tremendously powerful genetic tools have been ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes such as development are affected. The Caenorhabditis elegans germline is a great model system for studying PPIs that are related to the regulation of stem cells, meiosis, and development. There are a variety of well-developed techniques that allow proteins of interest to be tagged for recognition by standard antibodies, making this system advantageous for proximity ligation assay (PLA) reactions. As a result, the PLA is able to show where PPIs occur in a spatial and temporal manner in germlines more effectively than alternative approaches. Described here is a protocol for the application and quantification of this technology to probe PPIs in the C. elegans germline.

In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay

This protocol demonstrates use of the proximity ligation assay to probe for protein-protein interactions in situ in the C. elegans germline.

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...