The overall goal of this procedure is to show how to prepare primary cells dissociated from drosophila embryos for RNAi and cell imaging analysis. This is accomplished by first preparing fly cages. Synchronous embryos are then collected, washed and homogenized to dissociate the cells.
The primary cells are then plated and transfected with RNAi constructs. Ultimately, results can be obtained through immunofluorescence microscopy that show the effects of RNAi on differentiated primary cells. My name is Namo.
I'm a professor in the Department of Genetics at Harvard Medical School and an investigator of the Overuse Medical Institute. So today, I, Jonathan, a fellow in the lab as well as a former pro fellow in the lab, are going to introduce to you the technique of of generating primary cells from oph gas or embryos. So the technique is important for as a number of I import for a number of applications in the cell and developmental biology and in particular, as Janu showed a few years ago, the methodology can be used to for genome wide AI screens in in primary muscle cells.
The main advantage of this technique over existing methods like the analysis of established immortalized cell lines, is that primary cell culture more accurately reflects the biology of the organism. Generally, individuals leading to this method will struggle because several steps cannot be simply learned by reading the per To prepare killed yeast paste first grind granule, live yeast to a fine powder with a coffee grinder, then place it in a beaker and autoclave using a 20 minute dry cycle. Immediately after autoclaving.
Add enough sterile hot water to the yeast to make a paste with the consistency of toothpaste. To prepare primary cells dissociated from fly embryos, grow a wild type strain of drosophila such as orgon R in fresh fly bottles transfer newly ELOs flies to large embryo collection cages or fly population cages. Next, to facilitate the collection of synchronous embryos, move the cages into a room at about 25 degrees Celsius and 65%humidity with a 12 hour light dark cycle, feed the flies killed.
Yeast paste streaked on molasses plates. Replace the food plates once a day for two to three days. On the day of primary cell preparation, feed the flies with fresh plates for a one to two hour pre lay period.
Just before the 12 hour dark cycle, replace the pre lay plates with fresh molasses plates. Streaked with a minimal amount of yeast paste. Discard the pre lay collection which contains asynchronous older embryos.
Collect embryos for a period of one to two hours. Remove the plates containing the relatively synchronous population of embryos and store at 25 degrees Celsius for five to six hours. At this time, embryos will be at the advanced gastro stage to collect embryos.
After the five to six hour incubation, use a wet paintbrush to loosen them from the plates. Then place them in a set of stacked sieves and rinse thoroughly to remove as much yeast and fly debris as possible. Allowing the embryos to collect into the bottom sieve.
Tip the sieve so that the embryos accumulate on one side and gently wash them off from the sieve into a decoration basket. Next, sterilize and coate the embryos by immersing them in 50%volume per volume. Bleach for five to 10 minutes while the embryos incubate.
Fill it down to homogenizer with room temperature. M three medium to arrive at a volume no greater than 0.5 milliliters of embryos per 40 milliliters of medium. Then rinse the embryos off the wall of the basket with 70%volume per volume.Ethanol.
Using autoclave distilled water, rinse the embryos thoroughly to remove the bleach and or ethanol. Next, submerge the rinsed embryos in M three medium. In a sterilized beaker, let the embryo soak for two to five minutes while the embryos incubate disassemble the dec decoration basket and blot the NYX mesh dry with sterilized paper towels.
After the embryos have soaked in M three medium, transfer them into the homogenizer with a sterilized paintbrush, homogenized gently with short strokes using a loose pestle and avoid pushing it to the bottom of the tube when all the embryos are suspended gently but firmly homogenized with eight full strokes. Do not twist the pestle as it moves up and down. Transfer the homogenate into a sterile 50 milliliter conical centrifuge tube, either by pouring or pipetting and cap the tube centrifuge for 10 minutes at 40 cheese at room temperature to pellet the tissue debris large cell clumps and fatel in membranes.
Transfer the snat containing the cells and yolk to a clean tube and repeat the centrifugation step for five minutes. Next, carefully transfer the supernatant by pouring or pipetting it into a clean tube and spin for 10 minutes at 360 Gs at room temperature to pellet the cells, discard the supernatant. Re suspend the cell pellet in 10 milliliters of serum free culture medium for RNAi experiments.
To estimate the cell density, count the viable cells using a hemo cytometer. Then again, pellet the cells at 360 GS stg. Re suspend the cells in serum free M three medium at a concentration of one to five times 10 of the six cells per milliliter and plate them at 1.7 to 2.5 times 10 of the fifth cells per centimeter squared into each well of a 3 84.
Well plate dispense five microliters of double stranded RNAs at a final concentration of about 250 nanograms per well. Using a multi-channel pipette dispense 10 microliters of one to four times 10 to the six cells per milliliter of primary cells in serum free M three medium into each well of the 3 84 well plate. Alternatively cells can be cultured on eight well glass chamber slides for confocal imaging.
Centrifuge the plates for one minute at 300 Gs at room temperature. Put the plates at 25 degrees Celsius for 22 hours or at 18 degrees Celsius for one to two days. Add an additional 30 microliters of serum containing M three culture medium to each.
Well centrifuge the plates for one minute at 300 Gs at room temperature. Incubate the primary cells for an additional five to seven days at 25 degrees Celsius in a humid chamber. Finally, image the cells either live or after immunofluorescence staining.
Under optimal conditions, cells and culture will look healthy. With a round morphology, the culture will have little cell debris and be 50 to 80%confluent. After the initial plating, the cells will undergo morphological changes and be fully differentiated after about five to eight days in culture at 25 degrees Celsius, this brightfield image shows an example of primary cells right after plating.
The majority of cells are round, intact and well separated 20 hours after plating. The primary cells already form clusters and muscles with a branch like morphology can be recognized at this stage age. In this phase, contrast image primary cells cultured for five days look much more advanced with neuronal extensions, neuronal clusters, and muscles.
In these examples, primary cells were isolated from embryos carrying the DMF two GAL four D 42 G four, and UAS mito GFP transgenes in which DMF two G four and D 42 G four drive expression of MIT GFP in muscles and motor neurons respectively, the cells were treated with double stranded RNAs targeting either LA C or inflated. This fluorescence image shows both differentiated primary muscles and neurons that are expressing GFP in the culture transfected with controlled double stranded RNAs targeting LA C, the white arrow points to neuronal extensions while the arrow head points to a branch like muscle structure. When the differentiated muscle is stained with Phin, more than half of the muscles show a branch like structure while the rest show a rounded up morphology.
In the merged image, the muscles are stained yellow and the neuronal structures are stained green. This fluorescence image shows both differentiated primary muscles and neurons that are expressing GFP in the culture containing doublet stranded RNAs targeting inflated or if the white arrow points to neuronal extensions while the arrow head points to muscle when visualized with phin staining. Unlike the wild type controls that have some branch like muscles, all the muscles here are rounded up in this merged image, muscles are stained yellow while neuronal structures are in green.
Note that only a rounded up muscle morphology can be seen in this culture treated with if double stranded RNAs. While neuronal extensions look similar to those in wild type controls, these images show primary muscle cells that were fluorescently stained with anti drosophila titan in red and Phin that labels acton in green. These panels show wild type control muscle and these represent cells RNAi treated muscle, which developed into muscles with shortened sarcomere.
While attempting this procedure, it's important to remember to follow the protocol consistently in order to obtain comparable results across your experiments. Once mastered, this technique can be done within two to four hours if performed properly.
