The overall goal of this procedure is to examine micro tubial based organal transport in Drosophila S two cells and primary neurons. This is accomplished by first suspending Drosophila S two cells or drosophila neuroblast in cultured medium. The next steps are to plate the cells on con A coated cover slips and depolymerize the actin filaments so that there is no organelle transport on them.
Following this fluorescent markers are used to visualize the organelles of interest and time-lapse imaging is used to view organelle movement. Ultimately, results can quantify organelle motility through computational analysis. Our research focus is in characterizing micro dependent organal transport by using high-resolution microscopy inso cultured cells.
This video describes basic techniques of oph S two cells and brilliant neurons and tracking organal transport in these cells. Oph genetics allows us to examine gene function and primary neurons by loss of function mutations on the I or transgenic flies. This method was developed to extend our studies of augment transport from S two cells to primary neurons.
Begin by preparing the con. A coated cover slips. Place the cover slips in a ceramic rack and wash them by chromic acid immersion for an hour.
Next, rinse. The cover slips thoroughly with running water for 30 minutes until the acid is completely washed out. The cover slips should then be allowed to air dry.
Once dry coat the cover slips with con A for 30 minutes. Rinse the cover slips with distilled water for 15 minutes and let them air dry the con. A coated cover slips can then be stored at room temperature up to one month.
Grow up as two cells in T 25 or T 75 flasks depending on the number of cells required for the experiment. Count the cell density using a hemo cytometer. Place each con a cover slip into a 35 millimeter tissue culture dish.
Add one milliliter of growth medium. For example, insect express into a 35 millimeter tissue culture dish with con a coated cover slip and gently transfer approximately one times 10 to the fifth cells. The cell density is optimal for imaging because the processes of individual cells will not overlap immediately.
Add one milliliter of medium with five micromolar cyto, D cyto D depolymerizes, the actin filaments and induces process formation, which would occur after two hours at 25 degrees Celsius. To prepare a neuron culture, a serum supplemented Schneider's medium is needed for their growth. Store the supplemented medium at four degrees Celsius and warm it up to room temperature before its use.
Keep young adult flies in apple juice. Agar vials supplemented with dry yeast for one or two days before embryo collections for the neuron preparation to synchronize the collected embryos. Transfer the adult flies to a fresh apple juice agar vial for an hour in light before starting the collection.
Then transfer the flies to a new apple juice agar vial and discard the pre-collection vial. Transfer the vial to a dark place for embryo collection. After an hour, transfer the flies to another fresh vial for another hour.
Keep both of these collections in the dark to increase the embryo yield. Let the two vials of collected embryos develop for four more hours at room temperature. After four hours, the embryos will be at developmental stages nine to 11.
Collect at least 10 of these embryos per preparation. Quart some water into the vials and use a small paintbrush to loosen them from the agar surface. Now, rinse a transfer pipette in drosophila embryo wash solution.
Then use it to transfer the embryos in water to a 100 micron nylon mesh cell strainer. Clean up any agar or cotton fibers using a paintbrush or pipette tips. Next, fill a 1.5 milliliter micro tube with matching pestle with one milliliter of wash solution.
Then using the paintbrush, collect the embryos from the strainer into the micro tube. Now wash the embryos in embryo wash solution three times. Next, coate the embryos in a fresh one-to-one solution of commercial bleach and 95%ethanol for 10 minutes.
Put the con a coated cover slip into a 35 millimeter dish. Now sterilize the con A cover slips with UV light for 10 to 15 minutes. Dip the matching pestle into 70%ethanol for a few seconds and then air dry the pestle in the hood returning to the embryos under a hood.
Wash them twice with the supplemented Schneider's medium. Leave two to 300 microliters of medium in the tube with the embryos and gently mechanically dissociate the coated embryos. Using the pestle that matched the tube grind 10 to 15 times The level of embryo dissociation needs to be adjusted according to the embryo numbers.
For each preparation. Under dissociation will lead to clusters of cells that are difficult to image over. Dissociation will result in damage to the neurons and thus poor survival rate after overnight culture.
To remove the embryo debris centrifuge, the homogenized mixture at 20 G for two minutes, transfer the cells in the supernatant to a fresh tube. Pellet the cells by centrifugation at 550 G for two minutes. Resuspend the pellet in 500 microliters of supplemented medium.
Then repeat the centrifugation and resus once more, finish with Resus suspending the twice washed cells in 100 microliters of supplemented medium. Finally, plate the cells onto a sterilized con, a coated cover slip placed in a 35 millimeter dish. Incubate the cells for 10 minutes at room temperature to allow their attachment.
After 10 minutes, submerge the cover slips by adding two milliliters of supplemented Schneider's medium with five micromolar cyto D.Store the dish in a humidified 25 degrees Celsius incubator overnight before imaging drosophila S two cells attached on the con, A coated cover slip spread into flat round pancake shapes. However, when S two cells are plated on cover slips in the presence of cyto D and allowed to spread for two to three hours, they form long thin processes filled with parallel microtubules. These microtubules have uniform polarity with their plus ends out time-lapse fluorescence imaging of S two stably expressing m cherry tubulin shows that processes are very dynamic, especially during the first hour of attachment.
After an hour, growth slows down and after two to three hours the cells have fully grown processes. It is possible to visualize lysosomes transported along the micro tubial filled processes in the S two cells using lyo tracker in cells, stably transfected with peroxisome targeted GFP Peroxisomes were also seen moving along microtubules in primary cell culture from stage nine to 11 embryos. Neurons are a predominant cell type after overnight culturing.
They're easily recognized by their characteristic shape with 50 plus micron. Long neurites neuronal markers such as EL A are easily visualized. The processes in these neurons are filled with microtubules organelle transport can be tracked along the neurites.
As done for S two cells. Mitochondria were labeled with GFP under control of a motor neuron specific promoter peroxisomes were also labeled by peroxisome targeted SKLM cherry delivered by an injection of plasmid DNA into blast derm stage embryos. While attempting this procedure, it is important to remember to stage and dissociate the embryos correctly.
Once master, this technique can be done in two days if it is performed properly.
