The overall goal of the following experiment is to study the dynamics of ribonucleoprotein granules in primary cells, especially the formation of stress granules. This is achieved by first culturing cells like mouse, embryonic, fibroblasts, or hippocampal neurons from animals of interest. As a second step, a tagged component of the stress granules is transfected to allow their visualization.
Next, the cells are stress induced and monitored with time-lapse confocal microscopy. The results obtained show the dynamics of G three BP one containing stress granules in myths or mirror and hippocampal neurons based on their direct visualization. These method can help answer key questions about stress response in cells like neurons.
For example, in mice, knockdown of RNA, banning proteins essential for stress granules, neuronal defects were found. So this method can help understand the mechanisms behind these observation. After euthanizing an E 13.5 pregnant mouse, sanitize the mouse with 90%ethanol and remove the uterine horns under a sterile hood.
Transfer the horns to 37 degrees Celsius DPBS and remove the embryos. Transfer the embryos to a 100 millimeter dish. Remove the excess tissues and wash them off with warm DPBS.
Now under a microscope, remove the limbs, internal organs, and the upper part of the head containing the brain. The tail or head sections can be used for genotyping. Mince the remaining tissues in a separate dish for at least a minute.
Do not combine genotypes if they should vary within the litter. Cover the mince tissue with one x trypsin EDTA and incubate it for 30 minutes at 37 degrees Celsius. Next, add 10 milliliters of complete mes medium.
To stop the in reaction, loosen up the tissue with a pipette and transfer the resulting suspension to a 50 milliliter tube. Top the tube off with medium and allow the contents to settle. Then decant off the suspended cells.
Next, spin down the suspension at 300 Gs for five minutes. At room temperature, reus, suspend the pellet in six milliliters of complete medium per embryo. A count of viable nucleated cells should be around 5 million per embryo.
Then load 60 millimeter plates with three milliliters of cell suspension and culture.Them. Change the media the next day and allow the cells to grow to con fluency. When the cells are sub cultured, only fibroblasts will survive.
Split the confluent plates into 35 millimeter glass. Bottom dishes. Grow these dishes to between 50 and 70%co fluency and proceed with the transfection.
For each 35 millimeter dish, have three micrograms of purified plasmid for the transfection. Using a commercially available transfection kit will suffice. In this example, a vector expressing the RAs gap.
SSH three domain binding protein is transfected. It is an important component of stress granules. The procedure is of course, applicable to the study of any vector of interest in fibroblasts the day before harvesting the pup.
Prepare the Pauline lysine coated dishes begin by collecting embryos from 18.5 DPC dams and transferring them to HBSS in a large dish. If desired, neonatal pups can be substituted to prevent the sacrifice of the mother. Cut the head and remove the brain.
Then harvest the pup's brains to a clean dish with HBSS under a microscope. Remove all the meninges using fine forceps. Then separate out the desired parts of the brain such as the hippo campi.
Transfer the tissues to 4.5 milliliters of cold HPSS in 15 milliliter tubes and keep them on ice. Digest the tissues by adding 0.5 milliliters of 2.5%trypsin solution. Incubate them at 37 degrees Celsius for 15 to 20 minutes.
Then rinse the trypsin out with three washes of HBSS being careful not to lose any tissue. Now resuspend the tissues in one half to one milliliter of DMM with 10%FBS. Then homogenize the tissue by flowing it through the pipette.
Then switch to a 200 microliter tip and pipette the solution until there are no visible aggregates. Next, add 100 to 200 microliters of the cell suspension to 35 millimeter glass Bottom dishes prepared with DMM FBS media incubate the dishes. After three hours of culturing, replace the media with prewarm neuron complete media for the remaining culture time.
After five to 14 days in culture transfer the neurons using the calcium phosphate method. 24 hours after a successful transfection stress granules will begin assembling due to G three BP over expression. Added stress will further induce stress granule assembly, prewarm the confocal microscope components for at least 20 minutes, including warming the stage to 37 degrees Celsius.
A 35 millimeter plate holder is required just before visualizing the cells. Do the following. First, replace any cell medium containing phenol red with one without phenol red.
Start imaging the cells immediately. Stress granules will form in less than an hour, so stress will be induced after visualization of the cells. It is important to start the acquisitions as soon as possible after the induction of stress, and to precisely adjust two parameters depending on your cell type and your type protein.
The laser intensity and the duration of the acquisitions lower than both to reduce cytotoxicity. Next, view the cells with a 40 x and 63 x oil immersion objective. Make sure that the plate is flat in the holder and use the fluorescent light minimally.
Once a field of interest is located, use the software to acquire images. Start with the laser power at 10%or less, and set the gain and the offset to obtain the best possible signal to noise ratio. Scan at 1000 hertz to minimize exposure of the cells to the laser set disease stack as desired, and keep the scanning interval as long as possible to better maintain the culture's health.
Acquire data for as long as needed in MEFs granules form within 10 minutes and are clearly visible within an hour. In neurons, the process is a little slower tagged. G three BP should provide a reasonably strong signal for at least 45 minutes using the described techniques.
Stress granule formation was monitored in cultured cells. The localization of the G three BP tagged protein was diffuse in the cytoplasm cultured myths, and also diffuse in the cytoplasm of neurons. With arsonite treatment, the proteins clearly localize into defined and larger foci in either cell type.
These are the stress granules. The images taken can be reconstructed into a time-lapse movie. The first and second videos show fibroblasts responding to stress.
The third video shows the same. In a hippocampal neuron, Zack reconstructions offer 3D localization of the tagged G three B bp and thus a different perspective on tracking. Following this procedure, immuno cyto chemistry can be used to visualize the granule components after different kind of stress and after self fixation.
This can answer quantitative questions about the size and the number of the different cellular bodies in different conditions.
