Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

The overall goal of the following experiment is to measure the DNA damage and repair induced in mouse spleen cells by chronic in vivo exposure to low doses of beta or gamma radiation. This is achieved by first exposing the mice to internal beta or external gamma irradiation to trigger potential changes in DNA damage and repair. As a second step, the PLE cytes are isolated and irradiated with a high challenging dose of gamma radiation inducing detectable DNA damage to allow monitoring of the DNA repair.

Over time the spleen cells are then fluorescently immuno labeled against gamma H two A x to allow quantification of the DNA damage. Ultimately the DNA damage produced by doses of acute gamma radiation as low as 10 centigrade can be measured by flow cytometry. This method can help answer key questions in the radio protection field, such as do low doses of radiation typically encountered in an occupational, medical or public setting actually increase the probability of detrimental health effects such as cancer.

The main advantages of our technique over existing methods such as counting gamma H two A X foci using immunofluorescent microscopy are that our technique requires considerably less time for the analysis, making it suitable for large scale animal studies, and it reduces operator related bias in gamma H two A X quantification. The implications of this technique extend toward the personalization of medicine and radiotherapy of cancer and that individual patient radio sensitivities may be determined using peripheral blood lymphocytes to adjust the patient's dose regimen of the Radiotherapy. Though this method provides insight into the toxicity of radiation, it can also be applied to other studies such as investigating the toxicity of environmental chemical pollutants, or the fundamental mechanisms of DNA damage formation and repair in mice in vivo.

For internal beta irradiation, treat the animals for one month by AD libitum access to tritium water for external gamma irradiation. Place the entire cage of mice at the appropriate distance from a gamma radiation source to match the dose rate of the tritium exposure. Initiate and keep exposure for one month.

Use thermoluminescence dosimeters to measure the total absorb doses at the end of exposure. On the day of the spleen collection, place a cell strainer inside one small empty Petri dish per animal and add five milliliters of RPMI medium to each dish. Next place a euthanized mouse in the supine position and spray the hair with 70%ethanol when the animal has been fully soaked.

Use forceps to grab the skin anterior to the urethral opening and make a small incision in the perineal region from this incision. Cut along the ventral midline to the chest cavity, taking care to cut only the skin and not the muscle wall underneath. Dissect the skin away from the midline.

Then grasping the abdominal wall with forceps. Cut along the median access of the muscle to open up the abdominal cavity. Locate the spleen and lightly grip it with sterile forceps.

Then gently pull on the tissue while simultaneously cussing away the connective tissue. Remove about one 10th of the spleen for downstream analyses. Then transfer the rest of the tissue into one of the cell strainers for up to two hours after all of the spleens have been harvested.

Use sterile curved forceps to mince each of the tissues within their individual cell strainers. When the spleens have been sufficiently homogenized, remove the strainers and transfer the filtered cell suspension into 15 milliliter tubes. Rinse each of the strainers with an additional five milliliters of medium, pulling the washes with the rest of the cell suspensions and centrifuge the cells two times Resus, suspending the pellets in 10 milliliters of fresh RPMI each after the first spin.

Following the second spin. Re suspend the pellets in five milliliters of RPMI and transfer four milliliters of the resulting cell suspensions into 25 milliliter tissue culture flasks for incubation at 37 degrees Celsius and 5%carbon dioxide with 80%humidity. Transfer the remainder of the cell suspensions into new 1.5 milliliter tubes and centrifuge cells of four degrees Celsius.

Using a vacuum pump carefully aspirate the supernatants and gently resuspend the pellets in one milliliter of TBS buffer and spin down the cells again after vacuuming the supinate again. Reese suspend the pellets in 300 microliters of TBS each. Then while vortexing at low speed, at 700 microliters of minus 20 degrees Celsius, 100%ethanol to each tube.

Invert the tubes a few times to further mix. Then store the samples at minus 20 degrees Celsius for up to 12 months. To induce DNA double strand breaks for challenging the repair machinery.

Irradiator scy culture with two grays of gamma radiation at a dose rate of equal to or less than 200 mini gras per minute. Immediately following the challenge, return the culture to the CO2 incubator. One hour later, transfer the irradiated culture to a biological safety cabinet and use a pipette to gently resuspend the cells.

Transfer one milliliter of the resulting cell suspension to a 1.5 milliliter tube on ice, and then spin down the cells. Use the vacuum pump to carefully aspirate the supinate and then gently resus suspend the pellets in one milliliter of TBS. Then after a second TBS wash, fix the cells in ethanol as just demonstrated for minus 20 degrees Celsius storage for immunofluorescent analysis of the cells.

First, add 0.5 milliliters of ice cold TBS to the appropriate sample tubes. Next vortex aliquot Eloqua, a fixed minus 20 degrees Celsius stored SPL cytes for five seconds. Then transfer 0.5 milliliters of the cells into the appropriate tubes and spin them down gently re suspend the pellets in one milliliter of ice cold TBS containing 1%FBS and centrifugal cells.

Again, then incubate the samples in one milliliter of TST buffer per tube on ice for 20 minutes. After spinning down the cells, again, re suspend the pellets in 200 microliters of primary anti gamma H two ax antibody. Then position the tube on a rotating shaker at a 45 to 60 degree angle for 1.5 hours at 300 times G and room temperature at the end of the incubation.

Wash the samples two times in one milliliter of ice cold TBS containing 2%FBS. Then re suspend the pellets in 200 microliters of freshly prepared secondary antibody on the shaking platform. After one hour wash the cells in one milliliter of ice called TBS containing 1%FBS followed by a wash in one milliliter of ice called TBS alone.

Finally resuspend the pellets in 0.5 milliliters of TBS containing PROPIDIUM iodide In these graphs, representative flow cytometric results are shown The cells were first gated based on their electronic volume side. Scatter propidium iodine staining confirmed that both the control and radiation challenge samples exhibited a normal cell cycle distribution. While the measurement of the gamma H two ax signal demonstrated a greater than twofold increase in DNA double strand breakage within the two gray irradiated cells compared to the untreated controls here, relative gamma H two AX levels in mouse cytes, one hour after the ex vivo exposure to either low or high doses of gamma radiation are shown as expected.

A strong threefold induction of double strand breaks was detected after the two gray challenge. A slight but statistically significant increase was observed after exposure to 0.1 gray indicating a sufficient sensitivity of the method. The marked foci like pattern of fluorescence observed by microscopy indicates that the signal originates from the individual DNA double strand breaks within the CH chromatin validating the specificity of the staining.

Here, the level of DNA double strand breaks exhibited by PLE cytes harvested from mice exposed to one month of tritiated water are shown. Although the treatment resulted in a 10%increase in the basal gamma H two A X level, the change was not statistically significant. Further, no differences in the measured formation and loss of gamma H two ax.

After the challenge representing the kinetics of DNA double strand brake repair were observed between the cells from the tritiated water treated mice and the control. While attempting this procedure, it's important to remember to adhere to the rules and regulations established by your local radiation protection association and to utilize a certified laboratory or facility where large scale mouse projects can be carried out using both internal and external radiation sources. Once mastered, these techniques require only about one and a half days per 10 mice to complete if they're performed properly.

After watching this video, you should have a good understanding of how to use flow cytometric analysis of Gamma H two A X expression to measure DNA Doublet strand breaks and repair induced in vivo by exposure to low doses of tritium or gamma radiation. In addition to this procedure, other methods like M FISH in blood lymphocytes, the bone marrow MICRONUCLEUS assay and R-T-Q-P-C-R for measuring expression of stress response genes can be performed to evaluate the health risks associated with the exposure to low dose radiation.