In memory of Dr. M&#225;rcio Lorencini, a coauthor of this work, an excellent researcher in the &#64257;eld of cosmetics and devoted to promoting cosmetic research in Brazil. The authors are grateful for the Multi-user Laboratory in the Physiology Department (UFPR) for equipment availability and for the &#64257;nancial support of the Coordination for the Improvement of Higher Education Personnel (CAPES, Brazil) (Finance Code 001) and the Grupo Boticario.

NOTE: See the Table of Materials for the list of materials used in this protocol and Table 1 for the composition of solutions and media used in this protocol.
1. Preparing ZFL and ZEM2S cells
<ol>
	<li>Start with a T75 flask of ZFL or ZEM2S cells with 80% confluence, cultured in the respective complete medium at 28 &#176;C without CO2.</li>
	<li>Remove the culture medium from the flask and wash the cells by adding 10 mL of 1x phosphate-buffered saline (PBS) (0.01 M). Add 3 mL of 1x trypsin (0.05% v/v; 0.5 mM trypsin-EDTA) to the culture flasks. Incubate at 28 &#176;C for 3 min.</li>
	<li>Gently tap the flask to release the cells, and then stop the trypsin digestion by adding 3 mL of complete culture medium to the flask.</li>
	<li>Transfer the cell suspension to a 15 mL conical centrifuge tube and centrifuge at 100 &#215; g for 5 min.</li>
	<li>After centrifugation, carefully remove the supernatant, add 1 mL of complete medium for ZFL or ZEM2S cells, and resuspend the pellet using a micropipette.</li>
</ol>
2. Cell counting by trypan blue dye exclusion
<ol>
	<li>Add 10 &#181;L of the cell suspension and 10 &#181;L of trypan blue dye to a microtube to count the cells and evaluate their viability. Mix the cell suspension and dye using a pipette.</li>
	<li>Then, transfer 10 &#181;L of this mixture (cell suspension + trypan blue) to a Neubauer chamber and count the cells in the four large squares (Quadrants Q) placed at the corners of the chamber, considering viable cells to be those that do not take up trypan blue. Determine the number of viable cells using equation (1): 
	<img alt="Equation 1" src="/files/ftp_upload/64860/64860eq01.jpg" style="margin: auto;" /> (1)</li>
	<li>Calculate the final cell number in the cell suspension by multiplying the cell number determined using equation (1) by two (the dilution factor due to the use of trypan blue). 
	NOTE: Alternatively, an automated cell counting system (e.g., a cytomer with cell counting and viability function) can be used.</li>
</ol>
3. Cell plating in 96-well plates
<ol>
	<li>Calculate the cell suspension volume needed to obtain the number of cells required to perform the cytotoxicity assays. The number of viable cells for each cell line is indicated below:
	<ol>
		<li>Plate 60,000 viable ZEM2S cells per well; thus, for the entire plate, use six million cells in 20 mL of complete medium (200 &#181;L/well, 96-well plate).</li>
		<li>Plate 40,000 viable ZFL cells per well; thus, for the entire plate, use four million cells in 20 mL of complete medium (200 &#181;L/well, 96-well plate).</li>
	</ol>
	</li>
	<li>After that, transfer the respective volume of the cell suspension to a reagent reservoir (sterile) and fill up with the complete culture medium for ZFL or ZEM2S to 20 mL. Using a multichannel pipette, mix the solution gently up and down. 
	NOTE: Take care not to form foam or bubbles.</li>
	<li>Add 200 &#181;L of the cell suspension to each well of a transparent polystyrene 96-well plate using the multichannel micropipette. Incubate the plates at 28 &#176;C for 24 h. 
	&#8203;NOTE: The plate must have at least three wells without cells for the blank control, and only complete media should be added to these wells. The edge effect (caused by higher evaporation in the edge wells) commonly occurs in 96-well plate assays and can affect the viability of the cells in the edge wells of the plate28. This effect can be higher or lower depending on the 96-well plate brand and design28. Although we did not notice any cell growth/viability disturbance for ZFL and ZEM2S in the edge wells, we suggest sealing the plate with parafilm or adhesive sealing foil to prevent this effect, or culturing the cells only in the 60 inside wells and filling the edge wells with PBS.</li>
</ol>
4. Exposure of cells to test chemical
<ol>
	<li>Carefully discard the spent media from the wells using a multichannel micropipette.</li>
	<li>Expose the cells to test chemicals at different concentrations. Prepare the solutions of the test chemical concentrations in the culture media for ZFL or ZEM2S without fetal bovine serum (FBS) (exposure media). Then, add 100 &#181;L per well of these solutions in technical triplicate (i.e., three wells/test chemical concentration).</li>
	<li>For controls, place the control groups on the same plate as the test chemical in technical triplicates (three wells/control group). Thus, for the blank control (B), add 100 &#181;L of the exposure media in the cell-free wells, for the negative control (NC), add 100 &#181;L of the exposure media to wells with cells, and for the positive control (PC), expose the cells to a solution of 1% Triton X-100 prepared in the exposure media. In some cases, a solvent control (SC) should be included in the plate, considering a clearly non-cytotoxic concentration as a final solvent concentration. 
	NOTE: It is recommended to use 0.5% DMSO as solvent; DMSO can be used up to 1% as solvent in these cell lines without exceeding the cytotoxicity threshold of 10% related to the negative control.</li>
	<li>Incubate the plates at 28 &#176;C for 24 h. Seal the plates with parafilm or adhesive sealing foil to prevent culture medium evaporation. 
	&#8203;NOTE: Certain chemicals may have intrinsic background absorbance or fluorescence that may interfere with the absorbance or fluorescence of the indicator dye(s) (e.g., compounds with color may influence absorbance, serum albumin29, and compounds interfering with reduction enzymes30,31). In this case, the plate must include an additional control by adding test chemical solutions in the wells without cells. This is to verify the possible interference of the chemical auto-absorbance/autofluorescence with the dyes. If interference is detected, one should evaluate whether it can be excluded to obtain a correct prediction of cytotoxicity.</li>
</ol>
5. Cytotoxicity assays
NOTE: Prepare all solutions according to Table 1. All the steps described below (Figure 1) are carried out under sterile conditions. The use of a pipette to discard the exposure media is not recommended, because the cells can easily detach from the wells after chemical treatment.
<ol>
	<li>AB and CFDA-AM assays
	<ol>
		<li>After 24 h of test chemical exposure, carefully discard the exposure media by pouring the content into a collection tray.</li>
		<li>Wash the plate with 200 &#181;L of PBS. Carefully remove the PBS by pouring it into a collection tray to avoid losing cells.</li>
		<li>Add 100 &#181;L per well of AB/CFDA-AM solution. Incubate the plate for 30 min in the dark at 28 &#176;C.</li>
		<li>Measure the fluorescence in a fluorescence plate reader at 530 nm (excitation) and 595 nm (emission) for AB, and at 493 nm (excitation) and 541 nm (emission) for CFDA-AM.</li>
	</ol>
	</li>
	<li>NR assay 
	NOTE: The steps for the NR assay are carried out immediately after the AB and CFDA-AM assays (Figure 1).
	<ol>
		<li>Centrifuge the NR working solution (40 &#181;g/mL) at 600 &#215; g for 10 min. 
		NOTE: Precipitation of NR in the tube must not be transferred to the plates. Thus, after centrifugation of the NR working solution, collect the supernatant using a pipette without aspirating the NR precipitates. Transfer the supernatant to a reagent reservoir.</li>
		<li>Carefully remove the AB/CFDA-AM solution by pouring the content into a collection tray.</li>
		<li>Add 100 &#181;L per well of the NR working solution using a multichannel micropipette. Incubate the plate at 28 &#176;C for 3 h. 
		NOTE: After the 3 h incubation, observe if NR precipitation occurred in the plates using a microscope. NR precipitates may interfere with the quantification of the cell viability, thus, they should not be present.</li>
		<li>Carefully remove the NR solution by pouring the content into a collection tray. Wash the wells by adding 150 &#181;L of PBS per well.</li>
		<li>Add 150 &#181;L per well of the NR extraction solution and incubate the plate on a plate shaker for 10 min for gently shaking. Measure the absorbance at 540 nm in a plate reader. 
		NOTE: A second readout at 690 nm should be carried out to exclude any background fingerprint absorbance in the plate.</li>
	</ol>
	</li>
	<li>MTT assay 
	NOTE: The MTT assay must be carried out separately from the assays described above (in a new plate) (Figure 2).
	<ol>
		<li>Carefully remove the exposure media by pouring the content into a collection tray.</li>
		<li>Add 100 &#181;L of MTT working solution per well using a multichannel micropipette. Incubate the plate at 28 &#176;C for 4 h.</li>
		<li>Discard the MTT solution by pouring the content into a collection tray.</li>
		<li>Add 100 &#181;L per well of DMSO to extract the formazan crystals, incubating the plate on a plate shaker for 10 min. Measure the absorbance at 570 nm using a plate reader. 
		NOTE: A second readout at 690 nm should be carried out to exclude any background fingerprint absorbance in the plate. It is important to note that test chemicals may interfere with MTT, which must be evaluated to ensure the quality of the generated data32. For this, cell-free wells containing the test concentrations and MTT (0.5 mg/mL) should be exposed, followed by incubation to observe any color change in the wells that may increase the absorbance and lead to false viability results. Chemicals that interact with MTT must be avoided in this test.</li>
	</ol>
	</li>
</ol>
6. Calculating cell viability/cytotoxicity
NOTE: The raw absorbance or fluorescence acquired is used to calculate cell viability as a percentage related to the negative control (for test chemicals prepared directly in exposure media) or solvent control (for test chemicals prepared using solvents, such as DMSO). Before determining the cell viability percentage, the raw data must be normalized by the blank control.
<ol>
	<li>Calculate the average absorbance or fluorescence for each test chemical concentration and control group (three wells/treatment).</li>
	<li>To determine the cell viability percentage relative to control (negative or solvent), use equation (2): 
	<img alt="Equation 2" src="/files/ftp_upload/64860/64860eq02.jpg" style="margin: auto;" /> (2) 
	NOTE: Absorbance (abs) or fluorescence (fluo) units represent the mean of absorbance or fluorescence measured in the three wells per concentration; blank represents wells without cells.</li>
</ol>

The authors declare no conflict of interest.

Cytotoxicity assays are widely used for in vitro toxicity evaluation, and this protocol article presents four commonly used cytotoxicity assays modified to be performed in zebrafish cell lines (i.e., cell density for 96-well plate, incubation time in the MTT assay, FBS depletion during the chemical exposure condition, and maximal acceptable concentration for the SC). As these assays quantify cytotoxicity by different cell viability endpoints (metabolic function, lysosomal membrane integrity, and cell membrane in...

Chemical substances need to be tested regarding their safety for human health and the environment. Molecular and cellular biomarkers have been increasingly considered in safety assessments to predict effects on living organisms by regulatory agencies and/or legislations (e.g., REACH, OECD, US EPA)1,2, since they can precede the in vivo adverse outcome (e.g., endocrine disruption, immunological response, acute toxicity, phototoxicity)3,4,5,6,7. In this context, cytotoxicity has been taken as a measurement to predict fish acute toxicity5,8; however, it can have many other applications in ecotoxicity studies, such as defining sub-cytotoxic concentrations of chemical substances to study their most diverse set of effects on fish (e.g., endocrine-disrupting effects).
In cell culture systems (in vitro systems), the cytotoxicity of chemical substances can be determined by methods differing in the types of endpoints. For instance, a cytotoxicity method can be based on an endpoint related to specific morphology observed during the cell death process, while another can determine cytotoxicity by the measurement of cell death, viability and functionality, morphology, energy metabolism, and cell attachment and proliferation. Chemical substances can affect cell viability through different mechanisms, thus cytotoxicity assessment covering different cell viability endpoints is necessary to predict chemical effects9.
MTT and Alamar Blue (AB) are assays that determine effects on cell viability based on cell metabolic activity. The MTT assay evaluates the activity of the mitochondrial enzyme succinate dehydrogenase10. The reduction of yellowish 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) to formazan blue occurs only in viable cells, and its optical density is directly proportional to the number of viable cells10. The AB assay is a sensitive oxidation-reduction indicator, mediated by mitochondrial enzymes that fluoresce and change color upon reducing resazurin to resorufin by living cells11; however, cytosolic and microsomal enzymes also contribute to the reduction of AB and MTT12. These enzymes may include several reductases, such as alcohol and aldehyde oxidoreductases, NAD(P)H: quinone oxidoreductase, flavin reductase, NADH dehydrogenase, and cytochromes11.
The Neutral Red (NR) assay is a cell viability assay based on the incorporation of this dye into the lysosomes of viable cells13. The uptake of NR depends on the capacity of the cells to maintain pH gradients. The proton gradient inside the lysosomes maintains a pH lower than the cytoplasm. At normal physiological pH, the NR presents a net charge of approximately zero, which enables it to penetrate cell membranes. Thus, the dye becomes charged and is retained inside the lysosomes. Consequently, the greater the amount of retained NR, the greater the number of viable cells14. Chemical substances that damage the cell surface or lysosomal membranes impair the uptake of this dye.
The CFDA-AM assay is a fluorometric cell viability assay based on the retention of 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM)15. 5-CFDA-AM, an esterase substrate, is converted into carboxyfluorescein, a fluorescent substance that is polar and nonpermeable by membranes of living cells15; thus, it is retained in the inner side of an intact cell membrane, indicating viable cells.
Recently, three cytotoxicity assays (CFDA-AM, NR, and AB assays) were combined in a validated ISO (International Organization for Standardization) guideline (ISO 21115:2019)16 and OECD (Organization for Economic Co-operation and Development) test method (OECD TG 249) to evaluate fish acute toxicity using the RTgill-W1 cell line (permanent cell line from rainbow trout [Oncorhynchus mykiss] gill) in 24-well plates17. Although there is an existing cell-based method to predict fish acute toxicity, efforts have been invested in developing similar methods with other fish species and increasing the throughput of the method. Some examples include the development of ZFL cell lines transfected with reporter genes for specific toxicity pathways18,19, phototoxicity tests in the RTgill-W1 cell line20, and the use of ZFL and ZF4 cell lines (zebrafish fibroblastic derived from 1-day-old embryos) to assess toxicity by several cytotoxicity assays21.
Danio rerio (zebrafish) is one of the main fish species used in aquatic toxicity studies; thus, cell-based methods with zebrafish cell lines for fish toxicity testing may be extremely useful. The ZFL cell line is a zebrafish epithelial hepatocyte cell line that presents the main characteristics of liver parenchymal cells and can metabolize xenobiotics7,22,23,24,25. Meanwhile, the ZEM2S cell line is an embryonic zebrafish fibroblastic cell line derived from the blastula stage that can be used to investigate developmental effects on fish26,27. Thus, this protocol describes four cytotoxicity assays (MTT, AB, NR, and CFDA-AM assays), with modifications to be performed with ZFL and ZEM2S cell lines in 96-well plates.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-CFDA, AM (5-Carboxyfluorescein Diacetate, Acetoxymethyl Ester)</td>
			<td>Invitrogen</td>
			<td>C1345</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plate, 96 well plate</td>
			<td>Sarstedt</td>
			<td>83.3924</td>
			<td>Surface: Standard, flat base</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>12800-017</td>
			<td>Powder, high glucose, pyruvate</td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mix, powder</td>
			<td>Gibco</td>
			<td>21700026</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;s L-15 Medium</td>
			<td>Gibco</td>
			<td>41300021</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Neutral red&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>N4638</td>
			<td>Powder, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Orbital shaker&#160;</td>
			<td>Warmnest</td>
			<td>KLD-350-BI</td>
			<td>22 mm rotation diameter</td>
		</tr>
		<tr>
			<td>Dulbeccos PBS (10X) with calcium and magnesium</td>
			<td>Invitrogen</td>
			<td>14080055</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin sodium salt&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>R7017</td>
			<td>Powder, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Gibco</td>
			<td>31800-014</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>SFB - Fetal Bovine Serum, qualified, USDA-approved regions</td>
			<td>Gibco</td>
			<td>12657-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td>Powder,&#160; bioreagent for molecular biology</td>
		</tr>
		<tr>
			<td>Thiazolyl Blue Tetrazolium Bromide&#160; 98%</td>
			<td>Sigma-Aldrich</td>
			<td>M2128</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.5%), no phenol red</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>ZEM2S cell line</td>
			<td>ATCC</td>
			<td>CRL-2147</td>
			<td>This cell line was kindly donated by Professor Dr. Michael J. 
			Carvan (University of Wisconsin, Milwaukee, USA)</td>
		</tr>
		<tr>
			<td>ZFL cell line</td>
			<td>BCRJ</td>
			<td>256</td>
			<td></td>
		</tr>
	</tbody>
</table>

cytotoxicity assays with zebrafish cell lines

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute toxicity. Thus, this protocol presents cytotoxicity assays modified to evaluate cell viability in zebrafish (Danio rerio) embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates. The cytotoxicity endpoints evaluated are mitochondrial integrity (Alamar Blue [AB] and MTT assays), membrane integrity via esterase activity (CFDA-AM assay), and lysosomal membrane integrity (Neutral Red [NR] assay). After the exposure of the test substances in a 96-well plate, the cytotoxicity assays are performed; here, AB and CFDA-AM are carried out simultaneously, followed by NR on the same plate, while the MTT assay is performed on a separate plate. The readouts for these assays are taken by fluorescence for AB and CFDA-AM, and absorbance for MTT and NR. The cytotoxicity assays performed with these fish cell lines can be used to study the acute toxicity of chemical substances on fish.

Figure 3&#160;shows the plates of the AB, CFDA-AM, NR, and MTT assays. For the AB assay (Figure 3A), the blank wells and wells with no or a reduced number of viable cells show blue color and low fluorescence, while the wells with a high number of viable cells are pinkish and present high fluorescence values due to the transformation of resazurin (AB) into resorufin (pinkish substance) by the viable cells. For the CFDA-AM assay, there is no visible difference in ...

Watch this Scientific Journal Video about Cytotoxicity Assays with Zebrafish Cell Lines at JoVE.com

Cytotoxicity Assays with Zebrafish Cell Lines

This protocol presents commonly used cytotoxicity assays (Alamar Blue [AB], CFDA-AM, Neutral Red, and MTT assays) adapted for the assessment of cytotoxicity in zebrafish embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates.

Fish cell lines have become increasingly used in ecotoxicity studies, and cytotoxicity assays have been proposed as methods to predict fish acute ...

cytotoxicity-assays-with-zebrafish-cell-lines

Research

JoVE Journal

Environment

5.8K Views.  Federal University of Paraná (UFPR). This protocol presents commonly used cytotoxicity assays (Alamar Blue [AB], CFDA-AM, Neutral Red, and MTT assays) adapted for the assessment of cytotoxicity in zebrafish embryo (ZEM2S) and liver (ZFL) cell lines in 96-well plates.

Video: Cytotoxicity Assays with Zebrafish Cell Lines

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately $1.0 billion annually in the U.S.1 Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut2. However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually3-5. Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants.
Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

A Rapid and Efficient Method for Assessing Pathogenicity of Ustilago maydis on Maize and Teosinte Lines

The use of a needle injection method to inoculate maize and teosinte plants with the biotrophic pathogen Ustilago maydis is described. The needle injection inoculation method facilitates the controlled delivery of the fungal pathogen in between the plant leaves where the pathogen enters the plant through the formation of appresoria. This method is highly efficient, enabling reproducible inoculations with U. maydis.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is ...

A 3-dimensional (3D)-printed Template for High Throughput Zebrafish Embryo Arraying

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related research fields. Thanks to its unique features, including large fecundity, embryo translucency, rapid and simultaneous development, etc., zebrafish embryos are often used for large scale toxicity assessment of chemicals and drug/compound screening. A typical screening procedure involves adult zebrafish spawning, embryos selection, and arraying the embryos into multi-well plates. From there, embryos are subjected to exposure and the toxicity of chemical, or the effectiveness of the drugs/compounds can be evaluated relatively quickly based on phenotypic observations. Among these processes, embryos arraying is one of the most time-consuming and labor-intensive steps that limits the throughput level. In this protocol, we present an innovative approach that makes use of a 3D-printed arraying template coupled with vacuum manipulation to speed up this laborious step. The protocol herein describes the overall design of the arraying template, a detailed experimental setup and step-by-step procedure, followed by representative results. When implemented, this approach should prove beneficial in a variety of research applications using zebrafish embryos as testing subjects.

A 3-dimensional 3D-printed Template for High Throughput Zebrafish Embryo Arraying

Here, we present a protocol to design and fabricate a zebrafish embryo arraying template, followed by a detailed procedure on the use of such template for high throughput zebrafish embryo arraying into a 96-well plate.

The zebrafish is a globally recognized fresh water organism frequently used in developmental biology, environmental toxicology, and human disease related ...

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression.
The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified.
Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

The presented method creates natural herbivore damaged plant tissue through the application of Manduca sexta larvae to detached leaves of potato. The plant tissue is assayed for expression of six transcription factor homologs involved in early responses to insect herbivory.

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

Environmental DNA assays require rigorous design, testing, optimization and validation before the collection of field data can begin. Here, we present a protocol to take users through each step of designing a species-specific, probe-based qPCR assay for the detection and quantification of a target species DNA from environmental samples.

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The ...

Accumulation and Distribution of Fluorescent Microplastics in the Early Life Stages of Zebrafish

As a new type of environmental pollutant, microplastic has been widely found in the aquatic environment and poses a high threat to aquatic organisms. The bioaccumulation of microplastics plays a key role in their toxic effects; however, as a particulate, their bioaccumulations are different from many other pollutants. Described here is a feasible method to visually determine the accumulation and distribution of microplastics in zebrafish embryos or larvae using fluorescent microplastics. Embryos are exposed to different concentrations (0.1, 1, and 10 mg/L) of fluorescent microplastics with a diameter of 500 nm for 120 h. It is shown in the results that microplastics can bioaccumulate in zebrafish embryos/larvae in a concentration-dependent manner. Before hatching, strong fluorescence is found around the embryonic chorion; while in zebrafish larvae, the yolk sac, pericardium, and gastrointestinal tract are the main accumulated sites of microplastics. The results demonstrate the uptake and internalization of microplastics in zebrafish at early life stages, which will provide basis for better understanding the impact of microplastics on aquatic animals.

Zebrafish embryos/larvae develop externally and are optically transparent. The bioaccumulation of microplastics in fish at early life stages is readily assessed with fluorescently labeled microbeads.

As a new type of environmental pollutant, microplastic has been widely found in the aquatic environment and poses a high threat to aquatic organisms. The ...

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Here, we present an effective, easy, and fast 3D culture protocol for the formation of spheroids of two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte).

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known ...

Estimating the Integral Metabolite Cytotoxicity of Triazole Pesticides in Plants

Assessing Cytotoxicity of Metabolites of Typical Triazole Pesticides in Plants

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, causing potential threats to the ecosystem and human health throughout the food chain. The biotransformation of pollutants in plants generates a number of metabolites that may be more toxic than their parent compounds, implying that the metabolites should be taken into account during the toxicity assessment. However, the metabolites of pollutants in plants are extremely complex, making it difficult to comprehensively obtain the toxicological information of all metabolites. This study proposed a strategy to assess the integral cytotoxicity of pollutant metabolites in plants by treating them as a whole during toxicological tests. Triazole pesticides, a class of broad-spectrum fungicides, have been widely applied in agricultural production. Their residue pollution in farmland has drawn increasing attention. Hence, four triazole pesticides, including flusilazole, diniconazole, tebuconazole, and propiconazole, were selected as the tested pollutants. The metabolites were generated by the treatment of carrot callus with tested triazole pesticides. After treatment of 72 h, the metabolites of pesticides in carrot callus were extracted, followed by toxicological tests using the Caco-2 cell line. The results showed that the metabolites of tested pesticides in carrot callus did not significantly inhibit the viability of Caco-2 cells (P>0.05), demonstrating no cytotoxicity of pesticide metabolites. This proposed method opens a new avenue to assess the cytotoxicity of pollutant metabolites in plants, which is expected to provide valuable data for precise toxicity assessment.

Author Spotlight: A New and Efficient Method for Comprehensive Metabolite Cytotoxicity Assessment of Triazole Pesticides in Plants

The protocol describes a new method to assess the integral cytotoxicity of metabolites of triazole pesticides in plants.

Various organic pollutants have been released into the environment because of anthropogenic activities. These pollutants can be taken up by crop plants, ...