Name: cytotoxicity assays with zebrafish cell lines
Uploaded: 2023-01-06T14:30:00.000+00:00
Duration: 8 min 22 s
Description: Watch this Scientific Journal Video about Cytotoxicity Assays with Zebrafish Cell Lines at JoVE.com

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><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>FBS - Fetal Bovine Serum, qualified, USDA-approved regions</td><td>Gibco</td><td>12657-029</td><td></td></tr><tr><td>Ham&#039;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&#039;s L-15 Medium</td><td>Gibco</td><td>41300021</td><td>Powder</td></tr><tr><td>Neutral red&amp;nbsp;</td><td>Sigma-Aldrich</td><td>N4638</td><td>Powder, BioReagent, suitable for cell culture</td></tr><tr><td>Orbital shaker&amp;nbsp;</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&amp;nbsp;</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>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td>Powder,&amp;nbsp; bioreagent for molecular biology</td></tr><tr><td>Thiazolyl Blue Tetrazolium Bromide&amp;nbsp; 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.&lt;br/&gt; 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

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Environment

6.1K 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

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Cancer Research

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 ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

Developmental Biology

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

Immunology and Infection

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Biology

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Medicine

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Evaluating the Effects of Biotoxins on Immune Cell Functions in Zebrafish

A variety of biological toxins can be present at harmful levels in the aquatic environment. Cyanobacteria are a diverse group of prokaryotic microorganisms that produce cyanotoxins in the aquatic environment. These biotoxins can be hepatotoxins, dermatoxins, or neurotoxins and can affect fish and mammals. At high levels, these compounds are fatal. At non-lethal levels, they act insidiously and affect immune cell functions. Algae-produced biotoxins include microcystin and anatoxin A. Aquatic animals can also ingest material contaminated with botulinum neurotoxin E (BoNT/E) produced by Clostridium botulinum, also resulting in death or decreased immune functions. Zebrafish can be used to study how toxins affect immune cell functions. In these studies, toxin exposures can be performed in vivo or in vitro. In vivo studies expose the zebrafish to the toxin, and then the cells are isolated. This method demonstrates how the tissue environment can influence leukocyte function. The in vitro studies isolate the cells first, and then expose them to the toxin in culture wells. The leukocytes are obtained by kidney marrow extraction, followed by density gradient centrifugation. How leukocytes internalize pathogens is determined by endocytic mechanisms. Flow cytometry phagocytosis assays demonstrate if endocytic mechanisms have been altered by toxin exposure. Studies using isolated leukocytes to determine how toxins cause immune dysfunction are lacking. The procedures described in this article will enable laboratories to use zebrafish to study the mechanisms that are impacted when an environmental toxin decreases endocytic functions of immune cells.

This protocol describes flow cytometric assays that can evaluate the effects of toxin exposure on the endocytic functions of different subpopulations of zebrafish leukocytes. The use of specific functional inhibitors in the assay allows differentiation of the altered endocytic mechanisms.

A variety of biological toxins can be present at harmful levels in the aquatic environment. Cyanobacteria are a diverse group of prokaryotic ...

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage&#160;of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between&#160;3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.

This protocol describes a drug screening procedure in zebrafish larvae 3-7 days post fertilization followed by morphological assessment.

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...