The overall goal of this procedure is to use fluorescence to quantify the respiratory burst response. First, load the zebrafish embryos into the wells of a 96 well plate. Then add a fluorescent substrate and activate the respiratory burst with a chemical agent.
Then quantify the relative fluorescence using a fluorimeter. Ultimately, the fluorescence is used to show the respiratory burst potential. This method can be used to study how experimental manipulations affect overall innate immune health.
Here we use it to provide insight into the respiratory burst of zebrafish embryos, but it can also be applied to other systems such as adult zebrafish, kidney cells, and cell preparations from other fish species. Grow the embryos in deep Petri dishes at 28 degrees Celsius in egg water until the desired developmental stage. Remove any dead embryos daily.
Carefully decant the old egg water and replenish with new egg water daily. On the day of the experiment, make a reduced fluorescein working solution in a foil wrapped 1.7 milliliter micro centrifuge tube. Also prepare a working solution of 20 micrograms per milliliter PMA final concentration.
Then make five milliliters of each dosing solution in 15 milliliter conical tubes as described in the accompanying text. Keep the dosing solutions on ice first power on the Microplate reader and warm up the light source to measure the fluorescence. Set up a program detailing excitation and emission wavelengths, optics position sensitivity, and also include a five second shaking step.
Prior to the reed, arrange the experimental supplies of dishes with coated embryos. A black 96 well microplate a P 200 pipetter and tips an ice bucket with the fluorescein containing reagents. A multichannel P 200 pipetter, two sterile reservoirs, aluminum foil, and scissors.
Now cut a pipette tip such that the embryos or kidneys can fit through the opening. Set a P 200 pipetter to 100 microliters and transfer one embryo along with egg water into as many of the wells of a black 96 well microplate as desired. Now pour the dosing solution into a sterile 25 milliliter reservoir with a multichannel P 200 pipetter.
Add 100 microliters of H 2D CFDA dosing solution into one column of the 96. Well microplate repeat for half of the control embryo samples and half of the experimentally manipulated embryo samples. Pour the PMA containing dosing solution into a new sterile 25 milliliter reservoir.
Then aliquot this dosing solution into the remaining columns of the 96 Well microplate. Cover the Microplate with aluminum foil and place on a shaker for approximately 20 seconds. At 150 RPM, incubate the Microplate at 28 degrees Celsius when it is not being read.
Read the microplate after the addition of PMA. Continue taking measurements every few minutes for the desired time interval, or incubate the foil wrapped microplate at 28 degrees Celsius until a fixed time point. Next, retrieve the embryos from the wells euthanize the embryos according to the institutional animal care and usage protocol.
Also, dispose of the microplate and other disposable materials in the Biohazardous waste painter. Decide on the time point at which you would like to compare fluorescence. Subtract the average un induced control group's fluorescence value from the individual PMA induced control group's flu fluorescence values.
Repeat this for the experimental group with and without PMA. Then store these normalized fluorescence values into columns, the control plus PMA group and the experimental plus PMA group. Next, calculate the means and standard deviations for the normalized fluorescence values of the control plus PMA group and the experimental plus PMA group.
Compare the normalized fluorescence values using an unpaired T-test to determine statistical significance. Now, graph the means of the control plus PMA group and the experimental plus PMA group with error bars reflecting the appropriate standard deviations record the level of significance on the graph and in the figure legend. This experiment compares the respiratory burst response in wild type zebrafish embryos of AB background at 48 hours and 72 hours post fertilization.
Raw fluorescence was measured four hours after the addition of PMA in the PMA induced groups. The raw fluorescence values were higher in the 72 HPF population. As indicated by the calculated means fluorescence values are the normalized to account for variability in background levels of fluorescence.
Results of this respiratory burst assay revealed that the 72 HPF zebrafish embryos are able to produce significantly more reactive oxygen species and therefore mount a more robust respiratory burst response than 48 HPF embryos after induction with PMA. After watching this video, you should have a good understanding of how to use fluorescence to quantify the respiratory burst potential of zebrafish embryos. Don't forget that working with PMA powder can be extremely hazardous, so use precautions such as wearing a face mask while weighing out PMA powder.
