The overall goal of this proximity ligation assay experiment is to visualize and quantify the formation of DNA damage response and repair protein complexes following the induction of DNA damage in suspension cell cultures. This method can help answer key questions in DNA damage repair such as spatial temporal organization and regulation and how diverse repair pathways respond to different types of DNA damage. The main advantages of this technique are that it's relatively simple, requires a low cell number, and does not require extensive processing, which may disrupt or alter protein-protein interactions.
Though this method can provide insight into DNA repair, protein complex formation in culture-suspension cells, it can also be applied in cultured adherent cells, frozen or paraffin embedded tissues, and even whole animals. The human BCR able-positive, b-cell acute lymphoblastic cell line, BV-173, is used in this study and cultured in IMDM with supplements at 37 degrees Celsius in an atmosphere of 5%CO2. Plate the cells at two times 10 to the sixth per milliliter in a six well plate at five milliliters per well.
To arrest the cells in the G1 phase of the cell cycle, add five micromolar of ammonium methanesulfonate to each well and incubate the plate overnight at 37 degrees Celsius in an atmosphere of 5%CO2. On the following day, add five micromolar of an ATM inhibitor to each of two wells and return the plate to the incubator for 30 minutes. Next, induce DNA damage by adding 50 nanograms per milliliter of NCS to one of the wells that was pretreated with the ATM inhibitor and to another well that was not pretreated.
Incubate for two hours. Prepare 1XPBS plus 10%BSA by layering five grams of fraction-5 BSA on top of 50 milliliters of 1XPBS in a beaker and let it sit at room temperature without shaking or stirring until the BSA is dissolved. Slowly the filter the PBS plus 10%BSA solution through 0.22 micrometer filter and keep on ice.
Harvest the cells transferring the contents of each well to a 15 milliliter tube. Wash the cells twice with ice cold 1XBPS. After counting the cells, resuspend at two times 10 to the sixth cells per milliliter in 1XBPS plus 10%BSA.
Keep the cells on ice. Prepare the microscopy slides for this procedure by carefully polishing them with lint-free tissue and degreasing them by placing a 96%ethanol in a Coplin jar for five minutes. Let the slides air dry for 10 minutes.
Assemble the microscopy slides into the cytospin cytology funnels with an area of six milliliters. Precoat the slides by pipetting 50 microliters of 1XPBS plus 10%BSA into each funnel and centrifuge for one minute at 500 RPM. To each funnel, add 100 microliters of the cell suspension and centrifuge at 500 RPM for five minutes.
Carefully disassemble the funnels and make sure not to touch the spots on the slides. Use a Pap pen liquid blocker with a five millimeter tip to draw a circle around each spot. Then add 50 microliters of 4%PFA fixation solution to each spot.
And incubate for 30 minutes at room temperature. After 30 minutes, wash the cells with 1XPBS in a Coplin jar at room temperature with gentle agitation. Wash three times in this manner for five minutes each time.
Dry the slides around the spots with a lint-free tissue and remove as much liquid from the spots as possible by tilting the slide and drying with lint-free tissue. Add 50 microliters of 0.25%Triton X in 1XPBS to each spot and incubate for 10 minutes at room temperature without agitation. Wash the slides in 50 to 60 milliliters of TBS-T in a Coplin jar at room temperature with gentle agitation.
Three times for five minutes each time. Prior to blocking, tap off TBS-T and dry the slides around the spots with a lint-free tissue. Remove as much liquid from the spot as possible.
Briefly vortex the blocking solution and then add one drop of about 40 microliters to each spot. Place the slides in a pre-warmed humidified chamber and incubate for one hour at 37 degree Celsius. Prepare the antibody cocktails.
Vortex and then pipette commercial antibody diluent into each microfuge tube. Add the goat anti ATM antibody at 1:1000. And the rabbit anti-phosphoserine-15 p53 antibody at 1:100.
For single antibody control experiments prepare antibody dilutions containing only the goat anti-ATM antibody and only the rabbit anti-phosphoserine-15 p53 antibody. At the completion of the one hour incubation tap off the blocking solution but take care not to let the cells dry out. Add 30 microliters of each antibody cocktail dilution and incubate in a humidified chamber at four degree Celsius overnight.
On the following day, wash the slides with 1X NC2 wash buffer A in a Coplin jar at room temperature with gentle agitation two times for five minutes each time. After the slides had been washed twice with wash buffer A tap off the wash buffer from the slides and add 30 microliters of the proximity ligation assay probe mixture to each spot. Incubate at 37 degree Celsius in a prewarmed humidified chamber for one hour.
Wash the slides with 50 to 60 milliliters of wash buffer A in a Coplin jar at room temperature with gentle agitation. Next prepare the ligation ligase solution on ice by adding one microliter of ligase to 39 microliters of ligation solution for each spot. Add 40 microliters of the ligation ligase solution to each spot.
Incubate at 37 degree Celsius in a pre-warmed humidified chamber for 30 minutes. Wash the slides with wash buffer A in a Coplin jar at room temperature with gentle agitation. Prepare the amplification polymerase mixture on ice.
First dilute the amplification stock solution one to five in water. And add 0.5 microliters of polymerase to 39.5 microliters of 1X amplification solution for each spot. Tap off the wash buffer and add 40 microliters of amplification polymerase mixture per spot.
Incubate at 37 degree Celsius in a prewarmed humidified chamber for 100 minutes. At the completion of the 100 minute incubation tap off the amplification polymerase mixture and wash the slides with 1X NC2 wash buffer B in a Coplin jar. At room temperature with gentle agitation two times for 10 minutes each.
After that, wash the slides in 0.01x wash buffer B for one minute. Tap off excess wash buffer B and dry the slides around the spots with a lint-free tissue. Remove as much liquid from the spot as possible.
Prepare DAPI containing mounting medium that will not overstain the nuclei by diluting DAPI containing mounting medium 1:5 in mounting medium without DAPI. Add 25 to 30 microliters of the diluted DAPI containing mounting medium to a 24 millimeter by 24 millimeter cover slip. Pick up the cover slip with the slide and apply slight pressure to ensure that there are no trapped air bubbles.
Seal the cover slip with nail polish and wait at least 15 minutes before imaging. Finally, image and analyze the slides using a confocal fluorescence microscope. The proximity ligation assay was used to investigate the interaction between ATM and phosphoserine 15 p53 in BV 173 human BCR-able positive cells arrested in G1 phase.
In contrast to cells that were not treated with NCS to induce DNA damage, the majority of cells treated with NCS had punctate signals exclusively in the nucleus with an average of seven signals per cell. This indicates that induction of DNA damage results in the interaction between ATM and phosphoserine 15 p53. Pre treatment of the cells with a specific ATM kinase inhibitor prior to induction of DNA damage diminish the number of signals to an average of two signals per cell confirming that phosphorylation of p53 at residue serine 15 was dependent on ATM kinase activity.
Single antibody control experiments in which either the anti ATM or the anti phosphoserine 15 p53 antibody was emitted did not yield any signals as expected. This figure presents the quantification and statistical analysis of the results which demonstrate and confirm the specificity of the proximity ligation assay technique on cytospin preparations of suspension cell cultures. Once mastered, this technique can be done in about five hours after an overnight incubation with a primary antibody if its performed properly.
While attempting this procedure, it is important to remember to carefully titrate the antibody by immunofluorescence staining of the cells of your interest prior to performing proximity ligation assay. After its development, this technique paved the way for researchers in cell biology to explore transient protein protein interactions that are difficult to assess using standard methods such as immunoprecipitations and FRET based imaging which often results in technical artifacts. After watching this video you should have a good understanding of how to perform proximity ligation assay on suspension cell cultures to visualize and quantify protein protein interaction in situ.
