The overall goal of the following experiment is to observe the effect of ACF one or SNF two H knockdown on E four or four induced cell death. This is achieved by generation of cell lines in which ACF one or SNF two H srna expression is conditionally activated by addition of doxycycline resulting in reduced levels of ACF one or SNF two H protein as a second step. Cells of the cell lines that have been obtained are treated with doxycycline to reduce ACF one or SNF two H expression or are left untreated.
Next E four or four is expressed in the cells with or without S-H-R-N-A resistant ACF one or SNF two H.In order to investigate the effect of ACF one or SNF two H on E four or four induced cell death results are obtained that show the impact of ACF one or SNF two H levels on E four or four toxicity based on counting of nucleo with apoptotic morphology using the JY assay. The main advantage of this technique over other methods such as transient transfection of RNAs, is that all cells express the S-H-R-N-A and the percentage of cells co expressing together with traditional plasmid is higher. This method provides insight into the mechanisms underlying if 4 0 4 induced cell death, but it can also be applied to the study of other protic proteins.
In addition to Ana Lafe, a researcher from my laboratory will be demonstrating parts of this procedure To begin the procedure for generating inducible cell lines plate T-Rex 2 9 3 cells at a density of around five times 10 to the six cells per 10 centimeter plate in eight milliliters of DMEM containing serum without tetracycline and with five micrograms milliliter of blaster aside in incubate the plates overnight at 37 degrees Celsius and 5%carbon dioxide on the following day. Replace the medium with eight milliliters of fresh medium prior to transfection. The plasmid for transfection is the P superior neo plus GFP plasmid in encoding ACF one or SNF two H-S-H-R-N-A, driven by a tetracycline inducible H one promoter, as well as the neomycin resistance gene fused the GFP and driven by a constitutive PGK promoter.
Add 10 micrograms of plasmid DNA to 500 microliters of 150 millimolar sodium chloride and vortex. Then add the jet PY reagent at two microliters per microgram of DNA to another tube with 500 microliters of 150 millimolar sodium chloride and vortex. Add the jet pie solution to the DNA and mix well by vortexing.
Incubate it room temperature for 15 minutes. After 15 minutes, gently pipette the DNA complexes onto the 10 centimeter plates containing 70 to 80%confluence. T-Rex 2 9 3 cells a mix on the following day.
Replace the medium with a selective medium in this example DMEM as before, containing 500 micrograms per milliliter of G four 18. For the next two weeks, monitor the cells. Replace the selective medium with a similar fresh medium every three to four days until colonies appear.
Identify colonies by eye and mark their location at the bottom of the plate using a colored marker. Verify under the microscope that the colonies are well separated. Colonies can be isolated once they're big enough to be visualized without a microscope.
For the success of this procedure, it is important to choose well separated colonies during colony isolation. In a sterile hood, aspirate the medium from the plate. Gently rinse with PBS and aspirate well all remaining liquid.
Add three microliters of 0.25%trips in EDTA to one colony on the plate by pet the trips in repeatedly until the cells detach from the plate. Transfer the cells to selective medium in a well in a 24 well plate. Repeat this for several other colonies on the plate.
Grow the cells at 37 degrees Celsius, 5%carbon dioxide until they fill the well. Then split the cells from each original colony into three wells each in a separate 12 well plate and incubate overnight on the following day. Replace the medium in one plate with medium containing one microgram per milliliter doxycycline from a stock solution prepared in double distilled water.
Replace the medium in a control plate with medium without doxycycline. Incubate the cells at 37 degrees Celsius, 5%carbon dioxide for 72 hours. Cells from one treated and one untreated well are then harvested for western blot analysis to determine the efficiency of ACF one or SNF two H knocked down a representative result is shown here.
This blot was stained with antibodies to SNF two H and alpha tubulin. The latter serving as a loading control two of the clones. Number four and number five showed a strong reduction in SNF two H upon doxycycline induction and are thus chosen for use in the knockdown experiment, which will be demonstrated in the next segment.
The untreated cells in the third plate will be used to expand the selected cell line and aliquots of those cells will subsequently be frozen. For further use to induce knockdown of ACF one or SNF two H plate cells in selective medium in 10 centimeter plates, add doxycycline at one microgram per milliliter to half the plates and incubate 37 degrees Celsius, 5%carbon dioxide for 48 to 72 hours. Each group of plates must provide the cells for 12 six centimeter plates, including two duplicates for the DPI assay for each point, as well as one plate for western blot analysis of each sample.
48 to 72 hours after induction trypsin nose the cells from each group of cells and after counting them plate 1.5 times 10 to the six cells per six centimeter plate in the same medium with or without doxycycline. Incubate the cells at 37 degrees Celsius, 5%carbon dioxide overnight on the following day, transfect the cells. As indicated.
Both the untreated and doxycycline treated cells are transfected in triplicate in four ways. One with an empty plasmid and a plasmid expressing GFP two with the empty plasmid as well as a vector expressing SHRA resistant ACF one GFP or SNF two HGFP three with a plasmid encoding E four or four and a plasmid expressing GFP and with the plasmid encoding E four, all four. And the vector expressing SHRA resistant ACF one GFP or SNF two HGFP following transfection incubates all plates at 37 degrees Celsius and 5%carbon dioxide overnight.
On the day following transfection of cells extract proteins from one plate per sample for Western blot analysis to determine that ACF one or SNF two H has been efficiently knocked down and that E four or four was expressed equally in the different samples. The remaining 16 plates will be used for the DPI assay. Aspirate the medium from the plates intended for the DPI assay and wash the cells gently with PBS In a chemical hood.
Add one milliliter of 4%para formaldehyde prepared in PBS to cover the cells incubate for 15 minutes at room temperature without shaking. Aspirate the para formaldehyde and wash with PBS shaking at room temperature for five minutes. Repeat the wash two more times for a total of three washes after the third PBS wash at 80%Ethanol kept at minus 20 degrees Celsius and incubate at minus 20 degrees Celsius for at least one hour.
Aspirate the ethanol and wash the cells twice with PBS shaking at room temperature for five minutes each time after that wash once for five minutes at room temperature with PBS containing 0.5%BSA and 0.05%between 20 or P-B-S-B-T. Also with shaking to prevent non-specific antibody binding. Block the cells in one milliliter of P-B-S-B-T buffer containing 10%goat serum for 20 minutes while shaking at room temperature.
Then wash the cells twice in P-B-S-B-T five minutes each wash. Incubate the cells with the primary antibody, an E four or four specific antibody in this case in one milliliter of P-B-S-B-T for one hour while shaking a room temperature. Then wash twice in P-B-S-B-T and once in PBS containing 0.1%BSA five minutes each wash.
Next, add to the cells one milliliter of PBS 0.1%BSA containing the appropriate secondary fluorescently labeled antibody and 0.5 micrograms per milliliter. Final concentration of DPI incubate for 40 minutes while shaking at room temperature in the dark. Finally, wash with PBS for five minutes.
Dry the well by aspiration and keep the plates upside down for an additional hour to overnight to achieve complete drying and cover with aluminum foil mount cover slides on the cells using flora Mount G solution. Keep the plates at four degrees Celsius in the dark until ready to count. The apoptotic nuclei cells that underwent the various treatments described in the protocol were fixed and stained with E four or four specific antibodies and DPI to visualize the nuclei of transfected cells.
This figure shows an example of cells expressing E four or four and GFP panel A shows control GFP protein expression alone, and panel B shows E four or four expression DAPI stain nuclei are shown in panel C and the merged images are shown in panel D.The white arrows mark, GFP and E.Four or four transfected cells containing nuclei with apoptotic morphology. Red arrows mark nuclei with irregular shapes that are not counted as apoptotic nuclei asterisk mark mitotic nuclei or nuclei that just divided the number of condensed or fragmented nuclei visualized by DAPI. Staining was counted to calculate the percentage of nuclei with apoptotic morphology within the transfected cell population to maintain optimal objectivity of the test.
Counting of apoptotic nucleo in the various samples was performed in a blind fashion where the person counting was not aware of the sample identity A representative result is shown in this graph. Higher percentages of nuclear abnormalities were observed in E four or four expressing cells confirming the T four or four induces cell death. The highest percentage of nuclear abnormalities was observed in E four or four expressing cells where levels of ACF one were reduced by SHR and a mediated knockdown indicating that ACF one knockdown enhances E four or four Inge cell death enhanced E four or four toxicity in cells.
Expressing low levels of ACF one did not result from an increase in E four or four levels as seen in the Western blot. While performing this procedure, it is important to remember to facilitate an advise counting of the apoptotic nucle stent with DPI and to apply strict criteria for the identification of this nucle. In addition to this procedure, other methods such as clon genic assays can be performed to validate the results of the DA pse Following this procedure.
Other methods like co immunoprecipitation assays can be performed in order to answer additional questions. For example, whether there is a physical interaction between the investigated proteins.
