This protocol is significant because it allows the knockdown and over expression of genes in a developing fish, and the quantitation of those downstream effects on blood cells. This protocol is quick, easy to perform, economical, and easy to automate with access to the proper instrumentation. Demonstrating this procedure will be Kristen Rueb, an undergraduate researcher in the laboratory.
Begin by transferring between five and 200, 48 hours post fertilization embryos into a plastic 10 centimeter Petri dish. Tilt the dish so the embryo sink to the bottom edge, and remove as much E3 medium from the dish as possible. Then add 500 microliters of dechorionation protease to the embryos.
After five minutes at room temperature, tip all of the embryos to the bottom edge of the plate again and gently tap the side of the dish, allowing the embryos to gently rub against the bottom of the plate to completely remove their chorions. Using a squeeze bottle add approximately 20 milliliters of E3 medium to dilute the protease, and allow the embryos to settle. Then decant the medium, and rinse the embryos three more times with fresh medium as just demonstrated to remove all traces of the protease.
To prepare the embryos for dissociation, use a P1000 pipette to transfer five to 10 embryos into one, 1.5 milliliter micro centrifuge tube, and aspirate the transferred E3 medium. Then add one milliliter of 10 millimolar DTT in E3 medium to the embryonic zebrafish, and place the microcentrifuge tube in a horizontal position for 30 minutes at room temperature to remove the mucus coating. For embryo dissociation, wash the DTT treated embryos three times with one milliliter of DPBS supplemented with calcium and magnesium per wash.
Before adding 500 microliters of DPBS supplemented with calcium and magnesium, and five microliters of five milligrams per milliliter, dissociation protease. Then incubate the samples at 37 degrees Celsius on a horizontal orbital shaker at 185 revolutions per minute for 60 minutes. Be sure to periodically check on the embryos, so you don't over digest them.
When a not completely homogeneous sample with some solid tissue present can be observed, triturate the embryos with a P1000 pipette until the sample is fully dissociated. To prepare the dissociated zebrafish embryonic cells for flow cytometry, transfer the entire cell sample onto the top reservoir of a five milliliter polystyrene round bottom tube, with a 35 micrometres cell strainer cap. Rinse the cap with four milliliters of PBS without calcium or magnesium, and pellet the cells by centrifugation.
Then resuspend the cells in 500 microliters of PBS without calcium and magnesium per tube. For flow cytometric analysis of the zebrafish embryonic cells, gently vortex the cell suspensions before adding a one-to-one thousand dilution of red dead cell stain to each tube. Within 30 minutes of applying the dead cell stain, empty the waste tank, fill the sheath tank with the appropriate solution, and start the fluidic system of the flow cytometer.
In the analysis software, draw five plots, and set the first plot to measure forward versus side scatter. Set the forward scatter to linear, and the side scatter to logarithmic. Set the second plot to measure forward scatter height versus forward scatter width, and the third plot to measure scatter height versus side scatter width.
Set the fourth plot to measure the red dead cell stain on the x-axis, and side scatter on the y-axis to allow the discrimination of live from dead cells. The fifth plot should be set to measure the fluorophore of choice. When all of the plots have been set, load the sample onto the cytometer and reduce the flow rate so that the cells do not run out to quickly.
Adjust the forward and side scatter settings so that the bulk of the cell population can be clearly observed, and draw a gate around the cell population. Label this gate cells. With the second dot plot gated on the cells, gate on forward scatter and the side scatter singlets to exclude doublets.
In the fourth plot, adjust the red dead cell stain settings so that there is a clearly negative population. Draw a gate around these cells and label this gate live cells. In the fifth plot, gate on the live cells and draw a gate around the positive cells, then run each sample collecting at least 25, 000 live cell events.
When all of the samples have been analyzed, follow the shutdown procedure to turn off the fluidics, refill the sheath tank and empty the waste tank. After analyzing the percentage of GFP positive red blood cells from each embryonic zebrafish sample, the average of all the control group can be calculated. Typically, the average is set as one and all of the percentages are calculated from this reference point.
In this representative analysis, it was determined that reducing the ISM1 transcript with a specific morpholino, reduce the number of GFP positive red blood cells present within 48 hours post fertilization embryos. Rescuing this reduction in ISM1 morpholino levels with exogenous mRNA, return the number of red blood cells to normal. Proper digestion on the samples is critical.
If you over-digest or under-digest, you will not get the correct results. If a fax machine is available, researchers can collect the blood cells by sorting for in vitro culture, RNA sequencing and quantitative RT-PCR.
