This method can help answer key questions in the system biology field, such as the regulating mechanism behind the mitotic network and the dynamic properties and functions of the cell cycle clock. The main advantage of this technique is that it will generate a large quantity of droplets with multiple cell cycles in each, providing us with a high-throughput framework for quantitative manipulation and analysis. To begin, discard batches of eggs that have unclear boundaries between animal and vegetal poles or irregular white spots on top of animal poles.
Next, select an appropriate batch of eggs from one female frog, and transfer the eggs to a 600-milliliter beaker. Pour out an excess of 0.2x MMR buffer, and gently add cysteine in 1x extract buffer to the eggs. After this, shake the beaker vigorously by hand to remove the jelly coats of the eggs.
Repeat this wash three times with a total of 800 milliliters of cysteine buffer or until the eggs aggregate and settle at the bottom of the beaker. After the jelly coat is removed, wash the eggs four times in one liter of 0.2x MMR solution. Use a glass pipette to pick up and discard the eggs that turn white.
Pour the MMR buffer out of the beaker, and add 200 milliliters of calcium ionophore solution into the eggs. Use a glass pipette to stir the eggs gently until they are activated, and remove the calcium ionophore solution. Leaving the eggs in calcium ionophore solution for too long will initiate apoptosis.
We stir the eggs for 1.5 minutes and check the activation efficiency. If they are not activated, resume stirring, and check more frequently until 90%activation is achieved. After the eggs settle at the bottom of the beaker, check the activation efficiency.
Well-activated eggs have approximately 25%of the animal pole and 75%of the vegetal pole. Next, wash the eggs twice with a total volume of 50 milliliters of extract buffer supplemented with protease inhibitors. Then, carefully transfer the eggs to a 0.4-milliliter snap-cap microtube.
Centrifuge the microtube for 60 seconds at 200 g and then at 600 g for 30 seconds to pack the eggs. Then, use a glass Pasteur pipette to remove the excess buffer on top of the eggs after each step. After this, crush the eggs at 15, 000 g for 10 minutes at four degrees Celsius.
Use a razor blade to cut the tubes to separate the three layers of crushed eggs and keep the crude cytoplasm in the middle layer. Then, transfer the crude cytoplasm to clean ultracentrifuge tubes. Supplement the cytoplasm with protease inhibitors and cytochalasin B at 10 micrograms per milliliter each.
Centrifuge the crude cytoplasm at 15, 000 g and four degrees Celsius for five minutes. Then, use a razor blade to cut the tube and keep the cytoplasm in the middle layer. Place the freshly prepared extracts on ice.
First, add securin-mCherry mRNA to the prepared extracts. Next, add 200 microliters of 2%PFPE-PEG fluorosurfactant to the extract mRNA mixture. Use a vortex mixer to generate droplets, adjusting the vortex speed and duration as needed.
Then, visually inspect the droplets to ensure they are uniform in size. Using a 200-microliter pipette, transfer the droplets floating on top and an equal volume of surfactant to a PCR tube. Dip a glass tube into the droplet layer, and wait until the droplets fill approximately 75%of the tube.
Then, push the tube into the surfactant layer, and fill the tube completely. Next, fill a glass-bottomed dish with mineral oil. Then, use fine tweezers to immerse the droplet-filled tube in the oil.
Use a glass pipette to remove any visible debris or leaked droplets. To avoid movement and leakage of droplets, the glass tube filled with droplets should be immersed underneath mineral oil carefully. We can either push the tube on both ends with balance or add a few drops of mineral oil on the ends of the tube simultaneously first.
After this, use an epifluorescence microscope equipped with a motorized xy stage to image the droplets. Record time-lapse videos in the bright field and multiple fluorescence channels every six to nine minutes until the droplets lose their activity. Using this protocol, mitotic oscillation in both simple, nuclear-free cells and complicated cells with nuclei were produced.
The nuclei-free droplets generated mitotic oscillation up to 30 undamped cycles over 92 hours. The ability of the mitotic oscillator to drive downstream mitotic events was also examined. The self-sustained mitotic oscillator drove the changes of nuclear morphology observed through the autonomous alteration of distinct cell-cycle phases.
While attempting this procedure, it's important to remember to mind the time when making the extract, as it is time-sensitive, and always keep the eggs and extract on ice. After its development, this technique paved the way for researchers in the field of system biology to explore fundamental principles of complex clock properties, like tunability and stochasticity in Xenopus laevis.
