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The overall goal of the following experiment is to generate a purified population of yeast zygotes. This is achieved by first expressing cytoplasmic GFP or M cherry tagged poly zones. In haplo cells of opposite mating type, the cells are grown to logarithmic phase, then combined and co incubated to allow formation of zygotes.

The mating mixture is then harvested and sorted by flow cytometry in recovery mode to isolate red green double positive cells. This subpopulation includes both pre zygotic and zygotic entities. To further purify the sample, the red and green double positive population is subjected to a second round of flow sorting.

Using purity mode. This procedure yields an average purity of 90%zygotes based on visual examination and quantification of the cells by fluorescent microscopy. The advantages of this technique over existing methods such as sedimentation based purification are the following.

First, this technique provides a convenient visual assessment of zygote purity and maturation. Second, in our hands, the average purity of the zygotic fraction is approximately 90%much higher than that. For existing methods, Our purification method can help answer key biological questions surrounding the haid to diploid transition in eukaryotic cells on a molecular level, there is considerable conservation between lower and higher eukaryotes.

Therefore, studying yeast zygotes should promote a general understanding of zygote biology. Arguably, zygotes can also be considered a model for progenitor or stem cell function Grow s cera, VCA, Matt A and Matt alpha BY 47 41. Haid cells by streaking frozen stalks of yeast onto solid agar growth medium and incubating the plates at either room temperature or 30 degrees Celsius until colonies are present on the day preceding zygote purification labeled two 50 milliliter conical tubes for culturing transformed tabloids as soluble GFP and RPL 25 M.Cherry inoculate one culture with transformed tabloids expressing soluble GFP in the cytoplasm, and one with transformed tabloids expressing a single ribosomal protein.

RPL 25 fused with m cherry. Grow the cultures with shaking overnight at 23 degrees Celsius to reach an A 600, not exceeding 1.0. Next to conduct a cross, begin by adjusting the optical density of each sample to 1.0 in fresh, medium, approximately equivalent to three times 10 to the seventh cells per milliliter.

Next, mix equal volumes of the two cultures in fresh medium, in a 50 milliliter conical tube. Vortex briefly three times, then vortex for 10 seconds. Once position a pipette tip one centimeter above the surface of a room.

Temperature CSM glucose plate. Start a timer to count down from 30 minutes and immediately distribute 40 microliter droplets of the mixed cultures along the periphery of the plate spanning the circumference. Allow the plates to dry on the benchtop with the lid off for about 30 minutes.

Once the liquid has been absorbed, cover the plates and leave them undisturbed at room temperature for 1.75 to 2.5 hours. The process of polarized growth creates the characteristic schmo like morphology. During the schmo phase, the two haid cells continue to grow towards each other until achieving cell cell contact.

Subsequent cell cell fusion results in formation of the zygote. The nascent zygote then reenters the mitotic cell cycle giving rise to its first diploid bud. Following the incubation, place the plates on ice and harvest the samples by washing individual dried droplet areas 10 to 20 times with 150 microliters of cold CSM glucose.

Collect the wash solution in 15 milliliter conical tubes on ice. Next, keeping the samples on ice probe sonicate each of the tubes 10 times at 60%amplitude After sonic verify in a phase microscope that few or no aggregates remain, 15 to 50%of the cells are expected to have formed zygotes. At this point, leave the tube of cells in a vertical position, undisturbed on ice for 10 minutes as a precaution to allow any remaining aggregates to settle.

Recover the top most 90%of the volume and filter it through a nylon mesh polypropylene tube with a strainer cap. In a typical experiment, 10 to the seventh cells are recovered from one plate and four milliliters of CSM. Glucose cell sorting is done using a Sony eyesight reflection model flow cytometer with a HAPS one with a 100 micron tip.

Begin by selecting the excitation lasers in the win list 3D software for GFP excitation. Use the blue Argonne ion 500 milliwatt laser at 488 nanometers for M cherry excitation. Use the 100 milliwatt laser at 561 nanometers in the HAPS control software.

Ensure that recovery mode is selected and the sheath pressure for sorting is set to 21.5 pounds per square inch with a droplet frequency of 43, 400 drops per second and a sample pressure of 20.5 pounds per square inch base the sorting parameters on a forward versus back scatter plot in order to gate on viable cells. Note that the Sony eyesight machine used here uniquely indicates the side scatter plot as back scatter, but this back scatter is functionally equivalent to side scatter. Establish an emission plot of M cherry versus GFP to divide the events into four quadrants.

Red, positive green, negative red positive green positive red negative green negative and red negative green positive. Use non fluorescent cells as a standard for determination of the red negative green negative quadrant. For the initial sort.

Select recovery mode and collect the samples into chilled micro centrifuge tubes containing 250 milliliters of cold CSM glucose. Following the collection. Sediment the cells for 10 seconds at top speed in a refrigerated micro following the spin, aspirate the flow solution and resus suspend them in 500 microliters of cold CSM glucose.

Since high enrichment is not achieved with a single sort, briefly probe sonicate the partially purified sample twice at 60%amplitude. Sort the cells again this time using the purity mode option gate on viable cells and establish an emission plot of M cherry versus GFP sediment, the purified cells, and resuspend the pellets in 0.5 to 1.0 milliliters of CSM glucose. After the spin transfer, 0.5 milliliters into each of the wells of 24 well plates, then shake the cells for 15 minutes.

In 24 well plates at 23 degrees Celsius to allow metabolic recovery. To prepare agros pads, bring a suspension of 1.5%agros in CSM glucose to a boil in a 15 milliliter conical tube. Vortex the solution briefly and then apply 0.3 milliliter samples of the agro solution to the surface of at least two to three standard glass Slides immediately cover the slides with a second slide.

Do not apply pressure. Allow the slides to cool for 10 minutes. If the aros pads will not be used immediately, place them in a humidified chamber at room temperature without removing the upper slide.

These can be stored for up to a day. Then gently remove the upper slide by sliding it horizontally. Taking care to leave a smooth surface and not to disturb the aros layer quickly within one to two minutes deposit one microliter aliquots of sedimented cells on the aros pads without touching the surface.

Immediately cover the sample with a square cover slip using a single edged razor blade trim away. Excess agros uses syringe to dispense petroleum jelly along the edges of the cover slip capture images using delta vision with a 100 x oil immersion objective without binning. An ideal field is one that shows a single layer of cells with ample space in between to distinguish individual cells image with 0.2 to 0.4 micron intervals once the imaging is complete.

Devolve Zacks using the soft works 5.0 0.0 program and process minimally with a standard deconvolution of 20 cycles. Adjust intensity of red versus green fluorescence to minimize any background signal in the DeVol images to assess expression of fluorescent proteins in the transformed cells, cells were analyzed by flow cytometry before and after performing a cross prior to the cross. Essentially, all M cherry transformants were red and all the GFP transformants were green.

Note that while the M cherry signal was less intense than the GFP signal, it was detected by both cytometry and fluorescence microscopy. The cells were sorted and the brightest cells, which appear in the R two quadrant were selected and cultured with the GFP transform for 2.5 hours as can be seen here. The red and green positive sample represented 8.89%of total population following the cross.

This was enriched to 52.4%following the first recovery sort, then further enriched with the second purity sort when 21 similar preparations of red positive green positive cells were examined by phase contrast microscopy. To assess the number of zygotes, the average purity was 90%The contaminants were either green or red haplos or pairs of haplos that were in contact to determine the kinetics of initial zygotic bud emergence, as well as the incidents and distribution of medial lateral and terminal zygotic bud locations. Haplos were crossed for either 1.75 hours or 2.5 hours sorted by flow cytometry and then examined by epi fluorescent microscopy.

When cells were harvested after 1.75 hours, more than 50%were unbuttered and medial. Lateral or terminal buds were equally represented after 2.5 hours. However, there were approximately equal numbers of zygotes in each of four categories, unbuttered with medial, lateral, or terminal buds.

The structure of zygotes appears to remain unchanged during purification. Note that the GFP enters the nucleus and can be found in the nucleus even when the nuclei have not fused. ES stand out as black non fluorescent spheres.

The end product of purification yields a diverse population at various stages of development. Zygotes labeled A, have not undergone fusion. Type B zygotes are unbuttered.

Type C have a small bud, type D have a larger bud, and type E zygotes are about to undergo Cytokinesis note also that many of the earlier zygotes still exhibit a non fluorescent medial division line and that the valar inheritance structure can often be seen to extend from the zygote into the bud. While attempting this procedure, it is important to include both the recovery sort and the subsequent purity sort. We found that the second sort is key for increasing zygotic purity for the final product.

After watching this video, you should have a good understanding of how to conduct a cross generate purified zygotes by flow cytometry, assess purity by microscopic methods, and how to mount and photograph zygotes using fluorescent deconvolution microscopy.