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The overall goal of this procedure is to measure the relative cell cycle phase and cell size of tissues with cell populations genetically manipulated and labeled with GFP or RFP in Drosophila. First set up genetic crosses to generate progeny containing cells labeled with the desired fluorescent proteins and with the desired genetic manipulations in a specific tissue or population of cells. Under study cleanly isolate the desired tissue from the progeny of the proper developmental stage, then dissociate the tissue to a single cell suspension and stain with a viable DNA dye to measure DNA content.

Analyze the stained dissociated samples by flow cytometry. Ultimately, the in vivo results measure changes in cell size and cell cycle phase caused by the genetic manipulations under study. The main advantage of this technique over the existing method of flow cytometry on live drosophila tissues using ultraviolet dyes is that the dye cycle violet dye is compatible with less expensive benchtop cytometers containing a violet 4 0 5 laser, which can be purchased on a small budget and maintained on a single lab scale.

This method can help answer key questions in the cell cycle field by providing information on cell cycle phase and cell size that is more detailed than just measuring overall tissue size and proliferation rate. Demonstrating the procedure will be Kagel and Dan Sun, graduate students from my laboratory and Olga Guko. Our technician Collect mature larvae at the third larval Instar at about 110 to 120 hours after egg laying for the larva dissections.

Accurate pupil staging is essential for studies during metamorphosis for this harvest white pre pupa at the beginning of metamorphosis if necessary, I mesa sealed Petri dish of pupi or a vial of larvae in a 37 degree Celsius water bath To activate the heat. Chuck flip base for lineage tracing clones. After unsealing the Petri dish, place it in an incubator for aging to the desired developmental stage for pupil dissection.

Gently grasp a pupa by the anterior tip of the pupil case where there is an air bubble to avoid damaging the pupa inside position the floating pupa for proper grasping. Then with micro scissors cleanly cut through the posterior tip of the pupa at a slight angle toward the dorsal part of the animal to release the internal pressure without forcing the hist fat into the desired tissues in the thorax and head cut carefully along the dorsal median, avoiding damaging the wings and the anterior eye brain complex wash to remove his fat. Then remove the pupil from the pupil case by grasping the posterior edge of the pupil epidermis with forceps and pulling it out of the pupil case.

Transfer the dissected pupa to a clean dissection dish. Poke a small hole in the cuticle anterior to the eye brain complex with forceps. Using a glass pasti pipette with a rubber bulb.

Gently wash PBS dissection solution through the partially dissected pupa to remove any remaining his ized fat and gut tissue. To obtain the pupil wings, hold the pupa down by the head with one forcep using a second forcep. Grasp the most posterior tip of the wing cuticle, pull and dislodge it from the side of the carcass.

Then cut or pull off the wing at the hinge to remove it from the body and put it in a pile away from the carcasses and other material to obtain the pupil eyes or brain. Wash the pupa until the eye brain complex becomes dislodged and free from the pupa. Using a forcep, gently pull the pupil eye away from the brain and save the desired tissue Using a lubricated past pipette, carefully transfer the dissected tissues.

Incubate the tubes with shaking at 23 degrees Celsius at 500 RPM for 70 minutes. Then vortex gently for five seconds at a medium speed and return the samples to the shaker for an additional 15 to 20 minutes. Vortex samples once more for five seconds at medium speed for brains and pupil eyes.

Older than 36 hours. A PF dissociate tissues in DNA stain solution at 23 degrees Celsius at 500 RPM for 45 to 60 minutes. Then one by one manually dissociate large chunks of tissue by transferring to a dissecting dish along with about 50 microliters of DNA stain solution.

Rapidly pipette up and down with a manually drawn past your pipette of approximately 100 to 150 micrometer opening. Pool the dissociated specimens in a five milliliter tube. Incubate an additional 30 minutes at 23 degrees Celsius with shaking at 500 RPM.

Visually verify that the samples are fully dissociated. Mindful that the acellular transparent pupil wing cuticle will not associate. Begin the fax analysis by loading the sample onto the cytometer.

Then select channels to detect forward scatter and side scatter indicating cell size and membrane complexity. To detect the D cycle violet staining of the DNA, you will need to measure VL one height, width and area from violet laser excitation. Also select the blue laser channel one area to measure GFP fluorescence.

For RFP analysis, we use the blue laser channel three. This channel on the attune is optimized for RFP detection. Set the gates in order to restrict analysis to the selected population and to limit debris and sell clumps from the analysis that generates stop plots with gate one, exclude debris and clumps in the plot of SSC versus FSC in the plot of VL one area versus VL one with use GA two to exclude unstained cells sub G one apoptotic cells and cell clumps based upon identification of singlets.

GA three encompasses cells within gates one and two that are GFP negative on the plot of fluorescence versus DNA content based on VE L one height. Next gait four encompasses cells within gates one and two that are GFP positive on the plot of fluorescence versus DNA content. One can also generate gates five and six from dot plots of cell size as measured by GFP versus forward scatter.

To examine cell cycle phasing, use the two histograms that plot DNA content on the x axis and cell count on the Y ais for the populations defined by gate three and gate four. For the cell size, histograms set populations to gates five and six and plot FSC on the x axis and counts on the Y axis when running samples set the recording to stop at 10, 000. GFP positive events also set the acquisition volume for 300 microliters.

The appropriate threshold and voltage settings are contained within the provided templates. Run the samples at standard or high sensitivity 100 microliters per minute and record. The recorded data files generated by the Attune software are in FCS format, which is compatible with most cell cycle modeling software.

This representative workspace containing four dot plots and two histograms shows a larva wing sample expressing GFP and Cyclin D in the posterior wing, driven by an Grailed gal four transgene. The first dot plot of cell size with gait one excludes debris and clumps. The second dot plot discriminates singlets with gait two to exclude unstained cells sub G 1D NA content and clumps in the plot of GFP versus DNA content.

The GFP negative and positive cells can be distinguished and two N and four NDNA content is evident based upon position along the x axis. This plot of cell size as measured by GFP versus forward scatter is used to generate gates five and six. The resulting analyses lead to histograms of DNA content versus counts for the GFP or RFP negative population as well as GFP or RFP positive cells.

Using the provided RFP template. Similar results are obtained with the same tissue type and expression pattern. For RFP, the provided templates and voltages are suitable for analysis of larval eyes, brains, and wings, as well as pupil eyes, brains, and wings.

In the attuned software. Using the autoscale feature for the cell cycle or cell size histograms allows determination of the global maximum for each population. These cell cycle profiles represent GFP positive and negative cells in larva eyes expressing GFP in the posterior.

Using the GMR GAL four U-A-S-G-F-P Transgenes, the overlay reveals the differences in cell cycle phasing of the posterior GFP expressing cells. The relative change in cell size between GFP positive and negative cells can also be plotted. This cell cycle profile was derived from pupil brains at 46 hours A PF.The GFP positive cells are lineage tracing clones expressing the G one S regulator, cyclin D and E two F, that disrupt quiescence and lead to S-phase entry.

This is in contrast to the non expressing GFP negative cells, which at this stage are normally arrested. In G one, the attune software generates user-defined regions on the histograms, delineating G one s and G two phases. A statistics table for each run determines the absolute counts of cells within the user-defined regions and percentages.

Alternatively, modeling software such as mod fit can be used to estimate percentages in each phase as well as apoptotic cells. After watching this video, you should have a good understanding of how to isolate and label several tissues from Drosophila for cell cycle analysis.