High Throughput

The overall goal of this high throughput resazurin-based fluorometric assay is to monitor the effect of genetic or pharmacological manipulations on NADH2 production, which is a measure of metabolic rate. This method can help us answer key questions in the obesity field, such as the affect of a given drug or gene on metabolic rate. Additionally, this assay can be applied to screen for potential drug-drug or drug-gene interactions in a whole animal system.

The main advantage of this assay, relative to oxygen consumption assays, is the high throughput and the accumulation of signal across time, which allows the user to detect small differences in metabolic rate. Demonstrating this procedure will be Caroline Foy, a graduate student in my laboratory. After preparing a 60X stock solution of E3 medium according to the text protocol, prepare 1X E3 medium by diluting 16.5 milliliters of stock and one liter of double distilled water.

Then add 100 microliters of 1%methylene blue. To make flame polish disposable pipette for egg and fish transfer, use scissors to cut a graduated disposable transfer pipette at the 0.25 milliliter graduation. Then, to remove the rough edges, use a Bunsen burner to flame polish the cut tip.

After setting up zebrafish crossings and collecting eggs, allow the eggs to settle to the bottom of the dish, and pour off the water. Use a fine-tip disposable transfer pipette to remove any remaining water. Then add back the E3 medium.

Incubate the embryonic fish at 28.5 degrees Celsius to four days post fertilization, or DPF. Twice each day use a flame polished graduated disposable transfer pipette to remove any unfertilized eggs or dead embryos. Then once each day pour off the E3 medium.

Use a fine-tip transfer pipette to remove any remaining medium, and replace with fresh E3 pre-warmed to 28.5 degrees Celsius. To carry out the energy expenditure assay, prepare the assay solution, following this table. Use a 0.22 micrometer filter to filter sterilize the solution, and warm to 28.5 degrees Celsius in a water bath for the assay.

In a petri dish, combine all viable zebrafish embryos from the clutch. After filter sterilizing 75 milliliters of E3 medium, use a fine-tip disposable transfer pipette to remove as much of the E3 medium from the petri dish as possible. Add back 20 milliliters of sterile E3.Then remove and replace the sterile E3 two more times.

Next, using a flame-polished graduated plastic disposable transfer pipette, transfer individual embryonic fish to the wells of a 96-well plate. Working with one column of the plate at a time, remove the E3 medium from the eight wells of the first column, taking care not to touch the embryos with the transfer pipette. Then add 300 microliters of assay solution to each well.

Repeat with all 12 columns of the plate. To include a drug treatment, after preparing a 100X solution of the desired compound, add three microliters to each of the treatment wells. Then add three microliters of vehicle control to the control wells of fish.

To ensure that the fluorescence response to the compound is a result of activity within the fish, set up drug and vehicle treatments to three blank wells each in a 96-well plate. Now, using a fluorescence plate reader, with excitation and emission wave lengths of 530 nanometers and 590 nanometers respectively, read the wells of the fish plate. Place the plate into a humidified incubator at 28.5 degrees Celsius.

Depending on the assay and the stability of the compound being tested, read the fluorescence again at a predetermined time point. To assess the relative change in fluorescence, calculate the average change in fluorescence for the control wells. Then divide the change in fluorescence of each well by the average change in fluorescence of the control wells.

Here is an example of using two columns of a 96-well plate. Column A wells include vehicle-treated control zebrafish, while column B wells include insulin-treated zebrafish. In table A, the fluorescence measured at time zero is shown.

Table B contains the fluorescence measured 24 hours later. In table C, the fluorescence at time zero has been subtracted from the fluorescence at 24 hours to assess the change in fluorescence. Next, calculate the average change in fluorescence in control wells.

Table D is the change in fluorescence of each individual well, divided by the average change in the control wells. You can see here that insulin treatment at 10 micromolar caused a 2.77 fold increase in the signal generated by zebrafish over 24 hours by a P value of less than 0.0001. As seen here, the assay solution does not change color or absolute fluorescence levels in the absence of embryos.

However, the assay is highly sensitive to small changes in metabolic rate within the well. This figure represents signals generated by embryonic tilapia after incubating for 24 hours. Pink wells are indicative of fish with a high metabolic rate.

Purple wells contain fish with moderate metabolic rates, and the blue wells represent a low metabolic rate. This also shows the versatility of this assay and application across teleost species. Because the NADH2 induced reduction of alamar blue is non-reversible, the signal accumulates with time, which allows for small changes to be amplified.

Shown here is the relative change in fluorescence induced by one to five fish at one, two and four hours. With each increase in the number of fish, the relative change in fluorescence increase significantly with time, and the greater the number of fish, the greater the magnitude in fluorescence change with time. Once mastered, this technique can be performed on up to 5, 000 embryonic fish per day.

While attempting this procedure, it's important to remember to take care to avoid injuring the embryonic fish. After performing this assay, other procedures like Nile red measurement of neutral lipids, or measurement of phagic drive through intake of fluorassay labeled paramecium can be performed. This allows us to compare the relationship between metabolic rate and lipid metabolism, or metabolic rate and phagic drive.

After its development, this technique paved the way for researchers in the field of energy metabolism and metabolic disease to study energy expenditure in zebrafish without limitations in throughput. After watching this video, you should have a good understanding of how to use the fluorometric reagent resazurin to measure NADH2 production in the zebrafish.