Cell Cycle-specific Measurement of γH2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The method provides information about cell cycle dependent treatment effects and allows and in depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. This method combines important endpoints from the DNA damage response including double-strand frag recognition and repair, cell cycle effects, and eventual cell death by apoptosis into one comprehensive assay. We have used this technique mainly to investigate specific treatment responses and side effects of radiochemotherapeutic treatments in clinical oncology.

The assay is very useful for correlative studies in the fields of radiobiology and radiotherapy, but it can also be applied to various other areas of oncology. After collecting the cells from the culture according to standard cell collection procedures, resuspend the pellet in one milliliter of PBS. And transfer the cells into two milliliters of fixation solution in a hood.

After a 10 minute incubation at room temperature, collect the cells by centrifugation and decant the supernatant. It is important to fix the cells as a single cell suspension, and to keep the fixation time constant for all of the samples. Give adherent cells enough time to detach to obtain nicely rounded cells for analysis.

Then loosen the pellet by tapping and resuspend the cells in three milliliters of 70%ethanol. To wash the cells before staining, sediment the cells by centrifugation, and loosen the pellet by tapping. Then resuspend the cells with two consecutive washes with three milliliters of washing solution and one wash with one milliliter of washing solution.

Discarding the supernatant between each wash. After the last wash, carefully aspirate the supernatant without disturbing the pellet and resuspend the lucent pellet in 100 microliters of the antibody cocktail of interest. After one hour at room temperature, collect the cells by centrifugation and carefully aspirate the supernatant without disturbing the pellet.

Then, resuspend the loosened pellet in 100 to 250 microliters of DNA staining solution and dispense the cells through the cell strainer cap of a sample tube with a 35 micrometer mesh pore size. To analyze the cells by flow cytometry, open the parameters tab in the cytometer window and select the parameters as indicated in the table. Next, open the worksheet window and create plots as indicated.

Load a control sample onto the cytometer and press the run button. Select the first tube in the browser window and click the tube name. Adjust the detector voltages for forward and side scatter and DAPI in the parameters tab of the inspector window.

Press the standby button to continue with the worksheet setup in the software. Use the polygon gate tool to define the cells'population in the forward scatter area versus the side scatter area plot and use the rectangle gate tool to define the single cells'population in the DAPI with versus DAPI area plot. Press control G to show the population hierarchy and click on the default gate names to rename them.

Subsequently, right click on all of the histograms to select show populations and single cells from the context menu. Acquire control and treated samples to optimize the detector voltages for the antibody-coupled fluorophores to cover the full dynamic range of signal. To measure the samples, press the run button on the cytometer and use the acquisition dashboard in the software to measure the samples.

Then select file, export, and experiments to select directory export in the dialog box to save the data as dot FCS files. To evaluate the data, drag and drop the dot FCS files into the sample browser of the flow cytometric analysis software. And use the polygon tool to define the cells'population in the forward versus side scatter area plot and to exclude debris from the analysis.

In the DAPI with versus DAPI area plot, use the rectangle tool to define the single cells'population and to exclude any cell doublets or clumps from the analysis. In the DAPI area histogram, use the bisector tool to distinguish single cells with a normal DNA content from apoptotic cells with degraded DNA. Select tool biology and cell cycle to open the cell cycle modeling tool and select Dean-Jett Fox to estimate the frequency of cells in the G one S and G two M phases.

Create G one S and G two and M phase gates in the DAPI with versus DAPI area plot of the cell cycle population to enable a cell cycle specific gamma H two A X measurement. Use the bisector tool to distinguish phosphohistone H three positive and negative cells in the Alexa 555 area histogram of the cell cycle population and to distinguish CAS base three positive and negative cells in the Alexa 647 area histogram of the single cells'population. Press control T to open the table editor and configure the table as indicated.

Then select To File to set the format and destination in the output section of the menu ribbon and export the data into a spread sheet. Carbon ions induce higher gamma H two AX peak levels in glioblastoma cells that decline more slowly and remain significantly elevated in 24 to 48 hours compared to photon radiation at the same physical dose. For both carbon ions and photon radiation, the gamma H two AX levels were highest in G one phase cells likely because DNA double strand break repair is limited to the pathway of nonhomologous end joining at this stage in the cell cycle.

In line with the higher double strand break induction rate and slower repair kinetics, carbon ions induce a stronger and longer lasting cell cycle arrest in G two phase and a higher rate of apoptosis than do photons. Depending on the strength of the treatment effect, the resolution of the different cell cycle phases can be challenging. In some cell lines, the DAPI concentration has a big impact on the quality of the cell cycle profile and needs to be adjusted.

Slides can be prepared from the remaining samples after flow cytometry to evaluate the number, size, and shape of gamma H two AX foci and to distinguish between focal and parnuclear gamma H two AX staining. Using our method, we could show that mesenchymal stem cells are relatively resistant against ionizing radiations and topoisomerase inhibition but sensitive to gliomyacin. As PFA fumes are toxic, always use a fume hood when preparing the fixation solution and during the fixation step.

Also remember to wear gloves when handling DAPI.