The overall goal of this workflow is to determine whether foci identified within the cytoplasm are bona fide stress granules which are RNA granules that play important roles in cell physiology. The main advantage of this workflow is that it tests for all the fundamental properties of stress granules. Though this workflow is presented for human osteosarcoma U-2 OS cells, it can also be applied to other types of cell lines and primary cells.
Generally, individuals new to this method will struggle because not all stress induced foci are stress granules. Our workflow comprises all experimentation necessary to distinguish them. I will be demonstrating this procedure with Anais Aulus.
We are post doctoral fellows in the lab of Paul Anderson and Pavel Ivanov. To begin, seed cells onto coverslips placed in 24 well plates as described in the text protocol. After overnight incubation, aspirate the culture media from wells B1 to B4 and from C1 to C4 of the 24 well plate.
Add 500 microliters of the medium supplemented with 100 micromolars sodium arsenite to wells B1 and B2, then add 500 microliters of the medium supplemented with 50 micromolar sodium arsenite to wells B3 and B4.Next, add 500 microliters of the medium containing 150 micromolar vinorelbine to wells C1 and C2.Finally, add 500 microliters of the medium supplemented with 125 micromolar of vinorelbine to wells C3 and C4.Incubate the plate for 30 minutes at 37 degrees Celsius. After 30 minutes of incubation, add to the cells in column two five microliters of one milligram per milliliter cycloheximide solution and to cells in column four, two microliters of 1.25 milligrams per milliliter puromycin. Gently shake the plate to mix the solutions and return the plate to the incubator for an additional 30 minutes of incubation.
To fix the cells, remove the media from the wells and wash the cells with approximately 250 microliters of PBS. Add approximately 250 microliters of a buffered 4%paraformaldehyde solution to fix the cells, making sure the top of the coverslip is completely covered. Then, leave the plate on a bench rocker at room temperature for 15 minutes.
Next, remove and properly discard the paraformaldehyde. Add approximately 250 microliters of ice cold methanol to permeabilize and flatten the cells. Incubate the plate for an additional five minutes at room temperature on a rocker.
Once the permeabilization is complete, discard the methanol and block the cells by applying approximately 250 microliters of a blocking buffer for one hour at room temperature. Then, prepare the primary antibody solution by adding 12 microliters of the antibodies against G3BP1 eIF4G and eIF3B to three milliliters of blocking buffer. Add 250 microliters of the antibody solution to each well of the 24 well plate.
Incubate the plate on a rocker for at least one hour at room temperature. After one hour of incubation, remove the antibody solution and wash the cells with approximately 250 microliters of PBS for five minutes. Next, add 12 microliters of Anti-Mouse Cy2, Anti-Rabbit Cy3 and Anti-Goat Cy5 antibodies together with three microliters of hooks dye to three milliliters of blocking buffer to prepare the secondary antibody solution.
Add 250 microliters of the secondary antibody solution to each well and cover the plate to protect the samples from light. Incubate the plate on a rocker at room temperature for one hour. When the incubation is completed, remove the antibody solution and wash the cells with PBS for five minutes.
Heat mounting medium in a 37 degree Celsius heat block for 10 minutes to reduce viscosity. Then using a precut 200 microliter pipette tip, place 25 microliters of the mounting medium onto a labeled glass slide. With fine forceps, transfer the coverslip from the well to the slide, inverting the top so that the cells face the mounting medium.
Use a clean P200 tip to press the coverslip down. Once all the coverslips are mounted, remove the excess mounting medium by pressing a lab tissue firmly against the slide. Afterwards, flush the slide with water.
And immediately blot it with lab tissue once again. Permeabilize the prefixed cells by incubating them with 250 microliters of ice cold methanol at room temperature for 10 minutes. Then, to the emptied wells of the plate add approximately 250 microliters of 70%ethanol and seal the plate using parafilm to prevent evaporation.
Incubate the plate at four degrees Celsius overnight. After the overnight incubation, remove the ethanol and rehydrate the cells by adding 500 microliters of 2XSSC. Incubate the coverslips for five minutes on a rocker at room temperature.
Then, discard the solution and add 500 microliters of SSC once more. Incubate the plate for an additional five minutes with gentle agitation. Next, layer the parafilm on the bottom of a hybridization oven and pipette 25 microliters of a hybridization buffer on top of it.
Transfer the coverslip from the 24 well plate, inverting the top so that the cells face the hybridization buffer. Incubate the coverslips at 42 degrees Celsius for 15 minutes. Pipette 25 microliters of a biotin oligo dT solution onto the parafilm in the hybridization oven.
Then, using forceps, pick up the coverslip and blot its edge against a lab tissue to remove excess hybridization buffer. Transfer the coverslip to the biotin oligo dT solution, making sure the cells are face down and incubate at 42 degrees Celsius for 60 minutes. Once the hybridization is completed, add approximately 500 microliters of prewarmed 2XSSC to the wells of the 24 well dish.
Then, using forceps, transfer the coverslip to the well and incubate it at 42 degrees Celsius for 10 minutes. After further staining the cells with antibody solution, view the specimens using a fluorescence microscope. Presented here are images of the untreated U-2 OS cells that do not contain any granules or foci stained with stress granule markers G3BP1, eIF4G, and eIF3B.
In U-2 OS cells treated with sodium arsenite or vinorelbine, the formation of cytoplasmic foci that contain G3BP1, eIF4G and eIF3B was induced. The co-localization of these markers was confirmed by line scan analysis performed for each channel at increased magnification followed by super imposition of the obtained graphs. Additionally, cytoplasmic foci identified in the treated cells contained mRNA as shown by poly A FISH.
The oligo dT signal co-localized with the G3BP1 signal confirming the identify of stress granules. Once mastered, this entire workflow can be done in three days if it's performed properly. After watching this video, you should have a good understanding of how to perform immunofluorescence and fluorescent hybridization.
Steps that are critical for the correct characterization and identification of stress granules. Don't forget that working with paraformaldehyde could be extremely hazardous. Precautions such as use of PPE and adequate ventilation should always be used while performing this procedure.
