The cytotoxicity assay protocol using zebrafish cell lines can answer questions regarding acute toxicity of chemicals on fish or help define the appropriate test concentrations for other alternative fish assays. The main advantage of this 96-well plate assay is combined different viability endpoints to determine cytotoxicity for zebrafish cells which is an important fish species in ecotoxicity studies. The protocol is very simple.
If you follow all the described steps correctly, one should not encounter any problem. This protocol helps to achieve no animal-based ecotoxicity studies evaluating effects in fish cell lines derived from different organs or life stage such as hepatocytes in embryonic cells. To begin plating the ZEM2S or ZFL cells, calculate the volume of the respective cell suspensions needed to obtain the desired number of cells for assays.
Fill a sterile reagent reservoir with the complete culture medium for the corresponding cell line and transfer the calculated volume of the cell suspension to the reservoir for a total volume of 20 milliliters. Using a multi-channel pipette, gently mix the solution up and down without forming any foam or bubbles. Then using the multi-channel micropipette, add 200 microliters of the cell suspension to each well of a transparent polystyrene 96-well plate to obtain a viable cell count of 60, 000 cells per well for the ZEM2S cells and 40, 000 cells per well for the ZFL cells.
Incubate the plate at 28 degrees Celsius for 24 hours. To expose either type of cells to different concentrations of the test chemicals, prepare the desired concentration of test chemical solutions in the culture media for the cell line of interest without fetal bovine serum or FBS. Post the 24-hour incubation, carefully discard the spent media from the wells using a multi-channel micropipette.
Then add 100 microliters of a particular test chemical solution per well in technical triplicates, which means three wells for each test chemical concentration. Place the control groups on the same plate as the test chemicals in technical triplicates. For the blank control, add 100 microliters of the exposure media in three cell-free wells.
For the negative control, add 100 microliters of the exposure media to three wells containing cells. For the positive control, expose three wells containing cells to a 1%Triton X-100 solution prepared in the exposure media. Seal the plates with parafilm or adhesive sealing foil to prevent culture medium evaporation and incubate the plates at 28 degrees Celsius for 24 hours.
After 24 hours of the test chemical exposure, carefully discard the exposure media from the wells by pouring the content into a collection tray and wash each well with 200 microliters of PBS. Then to remove the PBS without losing cells, carefully pour it into a collection tray. To perform the Alamar Blue or AB and CFDA-AM assays, add 100 microliters of the respective solution per well and incubate the plate for 30 minutes in the dark at 28 degrees Celsius.
Next, measure the fluorescence in a fluorescence plate reader at the suitable excitation and emission wavelengths mentioned in the text. To perform the neutral red or NR assay, take the NR working solution having a concentration of 40 micrograms per milliliter and centrifuge at 600 G for 10 minutes. Next, carefully remove the previously added AB and CFDA-AM solutions from the wells by pouring the content into a collection tray.
Using a multi-channel micropipette, add 100 microliters of the NR working solution per well and incubate the plate at 28 degrees Celsius for three hours. Once the incubation is over, carefully pour the NR solution from the plate into a collection tray, and wash the wells by adding 150 microliters of PBS per well. After washing, add 150 microliters of the NR extraction solution per well and incubate the plate by gently shaking it on a plate shaker for 10 minutes before measuring the absorbance at 540 nanometers using a plate reader.
To carry out the MTT assay, after the 24-hour test chemical exposure, carefully remove the exposure media by pouring the content into a collection tray and using a multi-channel micropipette, add 100 microliters of MTT working solution per well in the dark. Incubate the plate at 28 degrees Celsius for four hours in the dark. Then discard the MTT solution by pouring the content into a collection tray and add 100 microliters of DMSO into each well to extract the formazan crystals.
Incubate the plate on a plate shaker for 10 minutes before measuring the absorption at 517 nanometers using a plate reader. For the AP assay, the wells corresponding to blank, positive control, and highly cytotoxic concentrations of test chemicals showed blue color and low fluorescence indicating either the absence or a reduction of viable cells. In contrast, the wells corresponding to low cytotoxic concentrations of test chemical and negative control containing a high number of viable cells converted the resazurin into pink resorufin having higher fluorescence.
For NR assay, highly cytotoxic concentrations of the test chemicals and positive control are transparent or pale pink with low absorbance, while wells containing viable cells retaining the NR dye displayed dark pink color and high absorbance. Similarly, for MTT assay, wells with no viable cells are transparent, while those containing viable cells transform MTT into violet-colored formazan. Based on the calculated cell viability percentages, the half maximal inhibitor concentration or IC50 value was estimated for the different test chemicals in ZFL and ZEM2S cell lines.
No significant difference in cell viability was observed for cultures with and without FBS, indicating the suitability of culture media deprived of FBS for the chemical exposure period of 24 hours. MTT and NR assays showed no significant effect of varying DMSO concentrations from 0.1%to 1%on cell viability, indicating that these cell lines support the use of DMSO as solvent up to 1%Pay special attention while handling plated cells to avoid self-detachment during the whole procedure and carefully prepare the neutral red working solution to avoid any dye precipitation. This procedure can be applied to studies addressing several aspects of chemical effects on fish using in vitro models, and contribute to the progress of no animal-based ecotoxicity studies.
