The overall goal of gamma-H2AX and 53BP1 immunofluorescence microscopy is to analyze the formation and repair of DNA double-strand breaks in various tissues. This method can help answer key questions in cancer research, such as how genetic instability arises in tumor cells. The main advantage of this technique is that single DNA double-string breaks can be visualized, and that the repair processes can be analyzed.
Demonstrating the procedure will be Susanne Brendel, a technician of our laboratory. Begin the experiment by preparing solutions, starting with the anti-coagulant stock solution with 200 International Units of heparin per milliliter in 0.9 per cent sodium chloride. Fill each of the collection tubes with two milliliters of anticoagulant stock solution before withdrawal of the blood or bone marrow samples.
Next, prepare the 10X Lysis solution for red cells with 82.91 grams ammonium chloride, 7.91 grams ammonium bicarbonate, and two milliliters of 0.5 molar ethylenediamenetetracedic acid solution to a pH of 8.0 by dissolving the agents in double distilled water for a total volume of one liter. Then, prepare the fixation solution by adding 8.3 microliters of a one molar potassium hydroxide solution to 360 milligrams of paraformaldehyde, or PFA, in a microtiter tube. Fill the tube with phosphate buffered saline, or PBS, to the one milliliter mark of the tube and heat the tube in a heating block at 95 degrees Celsius to obtain one milliliter of a 36 per cent PFA solution.
Transfer the one milliliter of 36 per cent PFA solution to a 15 milliliter tube. And add eight milliliters of PBS to obtain a nine milliliter four per cent PFA stock solution. Aliquot the four per cent PFA stock solution and store it at negative 18 degrees Celsius until use for cell fixation.
Next, prepare the permeabilization solution by adding 50 microliters of Octoxynol 9 to 50 milliliters of PBS to obtain a solution of approximately 0.1 per cent Octoxynol 9. Finally, prepare five per cent and two per cent blocking solutions by mixing the protein blocking agent with PBS. To prepare the blood or bone marrow samples, retrieve the tubes filled with two milliliters of the anticoagulant stock solution.
Withdraw seven milliliters of blood or bone marrow per tube under sterile conditions by venepuncture or bone marrow puncture respectively. Store the samples over night at room temperature. After storage overnight, dilute the heparinized blood sample one to one with PBS and the heparinized bone marrow sample in a ratio of one to three with PBS.
Suspend the cells gently by drawing them in and out of the pipette two times. Next, add one volume of density gradient medium to a fresh tube. Gently layer the diluted blood or bone marrow on top of the density gradient medium and take care not to mix the two layers.
Centrifuge the tube for 30 minutes at room temperature and 400 G.Draw the upper plasma layer off with a pipette. Then, carefully harvest the mononuclear cells in the layer above the density gradient medium. Transfer the mononuclear cells into a fresh tube and add at least three volumes of PBS.
Suspend the cells gently by drawing them in and out of the pipette two times. Centrifuge the tube. After the spin, remove the supernatant.
Add 10 milliliters of four degrees Celsius 1x Lysis solution for red cells. Re-suspend the cells gently by drawing them in and out of the pipette two times and place the tube on ice for five minutes. Then, add 30 milliliters of PBS at four degrees Celsius and centrifuge the tube.
Use CD34 micro beads and cell separation columns for the isolation of CD34 positive cells. Prepare two cyto-spins with mononuclear cells of the patient samples by centrifugation of one times 10 the five cells for each preparation. Fix the cells with 200 microliters of four per cent PFA for 10 minutes.
After fixation, wash the cells gently three times with 30 milliliters of PBS for five minutes each on a shaker. Then, permeablize the cells with 200 microliters of 0.1 per cent Octoxynol 9 for 10 minutes. Wash the cells gently three times with 30 milliliters of five per cent blocking solution for five minutes each on a lab shaker.
Block the cells in 30 milliliters of fresh five per cent blocking solution for one hour. The fixation and permeablization of the cells with four per cent paraformaldehyde in 0.1 per cent Octoxynol 9 preserves the gamma-H2AX and 53BP1 foci distinctly. Incubate one preparation of the cells with a mouse monoclonal anti-gamma-H2AX antibody and the other preparation of the cells with a mouse monoclonal anti-gamma-H2AX antibody and a polyclonal rabbit anti-53BP1 antibody overnight at four degrees Celsius.
The use of proven anti-gamma-H2AX and anti-53BP1 antibodies is highly recommended. After incubation, wash the cells gently three times with 30 milliliters of two per cent blocking solution for each five minutes on a lab shaker. Next, incubate the first preparation of the cells with an Alexa 488 conjugated goat anti-mouse secondary antibody diluted one in 500 in two per cent blocking solution.
Incubate the second preparation of the cells with an Alexa 488 conjugated goat anti-mouse secondary antibody and an Alexa 555 conjugated donkey anti-rabbit secondary antibody diluted one in 500 into two per cent blocking solution. Wash the cells gently three times with 30 milliliters of PBS on a laboratory shaker. Remove the PBS and mount the cells with mounting medium.
Cautiously put a cover slip on top of the mounting medium so that no air bubbles are embedded. Wait at least three hours for the mounting medium to harden before analyzing the cells by fluorescence microscopy. Finally, analyze the gamma-H2AX and 53BP1 foci in the cell nuclei with a fluorescence microscope equipped with filters for DAPI, Alexa 488, and Cy3 during imaging at a 100x objected magnification.
Analysis of gamma-H2AX foci in cells is most accurate in the G0 G1 phase and the G2 phase when gamma-H2AX foci appear as distinct fluorescent dots as shown in this representative image of fibroblasts. In contrast, analysis of gamma-H2AX foci in cells during the S phase is complicated by dispersed pan-nuclear gamma-H2AX speckles caused by the replication process. Immunofluorescence staining of gamma-H2AX was used for the quantification of DNA double-strand breaks in irradiated tissues and elucidated that the levels of gamma-H2AX foci in irradiated lymphocytes are proportional to the irradiation dose.
Co-localization of gamma-H2AX and 53BP1 foci in CD34 positive cells of a patient with acute myeloid leukemia indicates promotion of 53BP1 mediated nonhomologous end-joining repair mechanisms at sites of DNA double strand breaks. After this watching this video, you should have a good understanding of how to perform gamma-H2AX and 53BP1 immunofluorescence microscopy for the analysis of DNA double strand breaks. Following this procedure, other methods like immunoblotting of phosphoryrelated ATM, ATR, checkpoint kinase one, checkpoint kinase two and TP53 can be performed in tumor cells in order to analyze alterations of the DNA damage response.
