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

Podsumowanie

The presented method combines the quantitative analysis of DNA double-strand breaks (DSBs), cell cycle distribution and apoptosis to enable cell cycle-specific evaluation of DSB induction and repair as well as the consequences of repair failure.

Streszczenie

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer treatments as used in clinical oncology. It enables a quantitative and longitudinal analysis of the DNA damage response after genotoxic stress, as induced by radiotherapy and a multitude of anti-cancer drugs. The method covers all stages of the DNA damage response, providing endpoints for induction and repair of DNA double-strand breaks (DSBs), cell cycle arrest and cell death by apoptosis in case of repair failure. Combining these measurements provides information about cell cycle-dependent treatment effects and thus allows an in-depth study of the interplay between cellular proliferation and coping mechanisms against DNA damage. As the effect of many cancer therapeutics including chemotherapeutic agents and ionizing radiation is limited to or strongly varies according to specific cell cycle phases, correlative analyses rely on a robust and feasible method to assess the treatment effects on the DNA in a cell cycle-specific manner. This is not possible with single-endpoint assays and an important advantage of the presented method. The method is not restricted to any particular cell line and has been thoroughly tested in a multitude of tumor and normal tissue cell lines. It can be widely applied as a comprehensive genotoxicity assay in many fields of oncology besides radio-oncology, including environmental risk factor assessment, drug screening and evaluation of genetic instability in tumor cells.

Wprowadzenie

The goal of oncology is to kill or to inactivate cancer cells without harming normal cells. Many therapies either directly or indirectly induce genotoxic stress in cancer cells, but also to some extend in normal cells. Chemotherapy or targeted drugs are often combined with radiotherapy to enhance the radiosensitivity of the irradiated tumor^1,2,3,4,5, which allows for a reduction of the radiation dose to minimize normal tissue damage.

Ionizing radiation and other genotoxic agents induce different kinds of DNA damage, including base modifications, strand crosslinks and single- or double-strand breaks. DNA double-strand breaks (DSBs) are the most serious DNA lesions and their induction is key to the cell killing effect of ionizing radiation and various cytostatic drugs in radiochemotherapy. DSBs do not only harm the integrity of the genome, but also promote the formation of mutations^6,7. Therefore, different DSB repair pathways, and mechanisms to eliminate irreparably damaged cells like apoptosis have developed during evolution. The entire DNA damage response (DDR) is regulated by a complex network of signaling pathways that reach from DNA damage recognition and cell cycle arrest to allow for DNA repair, to programmed cell death or inactivation in case of repair failure⁸.

The presented flow cytometric method has been developed to investigate the DDR after genotoxic stress in one comprehensive assay that covers DSB induction and repair, as well as consequences of repair failure. It combines the measurement of the widely applied DSB marker γH2AX with analysis of the cell cycle and induction of apoptosis, using classical subG1 analysis and more specific evaluation of caspase-3 activation.

The combination of these endpoints in one assay not only reduces time, labor and cost expenses, but also enables cell cycle-specific measurement of DSB induction and repair, as well as caspase-3 activation. Such analyses would not be possible with independently conducted assays, but they are highly relevant for a comprehensive understanding of the DNA damage response after genotoxic stress. Many anti-cancer drugs, such as cytostatic compounds, are directed against dividing cells and their efficiency is strongly dependent on the cell cycle stage. The availability of different DSB repair processes is also dependent on the cell cycle stage and pathway choice which is critical for the repair accuracy, and in turn determines the fate of the cell^9,10,11,12. In addition, cell cycle-specific measurement of DSB levels is more accurate than pooled analysis, because DSB levels are not only dependent on the dose of a genotoxic compound or radiation, but also on the DNA content of the cell.

The method has been used to compare the efficacy of different radiotherapies to overcome resistance mechanisms in glioblastoma¹³ and to dissect the interplay between ionizing radiation and targeted drugs in osteosarcoma^14,15 and atypical teratoid rhabdoid cancers¹⁶. Additionally, the described method has been widely used to analyze side effects of radio- and chemotherapy on mesenchymal stem cells^{17,18,19,20,21,22,23,24}, which are essential for the repair of treatment-induced normal tissue damage and have a potential application in regenerative medicine.

Protokół

1. Preparation

1. Prepare ≥1 x 10⁵ cells/sample in any type of culture vessel as starting material.
   1. For example, conduct a time-course experiment after exposure of U87 glioblastoma cells to ionizing radiation: Irradiate sub-confluent U87 cells in T25 flasks in triplicates for each time point. Choose early time-points (15 min up to 8 h after irradiation) to follow the kinetics of DSB repair (γH2AX level) and late time points (24 h up to 96 h) to assess residual DSB levels, cell cycle effects and apoptosis.
      NOTE: The protocol is not restricted to irradiation experiments or any specific cell line. It has been tested with numerous cell lines of all types, from different species and for various treatment conditions.
2. Prepare the following solutions including 10% excess volume.
   1. Prepare 2 mL per sample of fixation solution composed of 4.5% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Prepare the solution fresh. Dilute PFA in PBS by heating to 80 °C with slow stirring under the fume hood. Cover the flask with aluminum foil to prevent heat loss. Let the solution cool to room temperature and adjust the final volume. Pass the solution through a folded cellulose filter, grade 3hw (see the Table of Materials).
      CAUTION: PFA fumes are toxic. Perform this step under a fume hood and dispose PFA waste appropriately.
   2. Prepare 3 mL per sample of permeabilization solution composed of 70% ethanol in ice-cold H₂O. Store at -20 °C.
   3. Prepare 7 mL per sample of washing solution composed of 0.5% bovine serum albumin (BSA) in PBS.
   4. Prepare 100 µL per sample of 3% BSA in PBS as antibody diluent.
   5. Prepare 100-250 µL per sample of DNA staining solution composed of 1 μg/mL 4',6-diamidin-2-phenylindol (DAPI) in PBS.
3. Set the centrifuge for 15 mL tubes to 5 min at 200 x g and 7 °C. Let the centrifuge cool down and use these settings for all centrifugation steps.

2. Sample Collection

1. If processing adherent cells (e.g., U87 glioblastoma cells grown in T25 flasks with 5 mL of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C and 5% carbon dioxide atmosphere), continue with steps 2.1.1. and 2.1.2. For suspension cells proceed directly to step 2.1.2.
   1. Collect the medium in a centrifugation tube. Detach the cells using a routine cell culture method, which may include the use of trypsin, ethylenediaminetetraacetic acid (EDTA) or other cell detachment agents.
      1. For U87 cells, prewarm PBS and trypsin/EDTA (see the Table of Materials) to 37 °C, wash the cell layer with 1 mL of PBS, incubate the cells for 1-2 min with 1 mL of trypsin/EDTA and support cell detachment by tapping at the flask. Collect all washing solution and the cell suspension in the tube with the medium.
   2. Centrifuge the cells, discard the medium and resuspend the cells in 1 mL of PBS.
2. Pipet the cells up and down several times to ensure a single cell suspension and transfer the cell suspension into a tube with 2 mL of fixation solution (4.5% PFA/PBS, 3% final concentration).
   CAUTION: PFA fumes are toxic. Perform this step under a fume hood and dispose PFA waste appropriately.
3. Incubate the cells for 10 min at room temperature.
4. Centrifuge the cells and discard the supernatant by decantation.
5. Loosen the cell pellet by tapping onto the tube and resuspend the cells in 3 mL of 70% ethanol. Proceed directly with the next step or store the samples at 4 °C for up to several weeks.

3. Washing and Staining

1. Centrifuge the cells and discard the supernatant by decantation.
2. Loosen the cell pellet by tapping onto the tube. Resuspend the cells in 3 mL of washing solution (0.5% BSA/PBS), centrifuge and discard the supernatant.
3. Repeat the washing step 1x with 3 mL, and then 1x with 1 mL washing solution. In the last step, discard the supernatant carefully by pipetting. Take care not to aspirate the pellet.
4. Dilute the antibodies against γH2AX, phospho-histone H3 (Ser10) and caspase-3 (see the Table of Materials) in 100 µL/sample with antibody diluent (3% BSA/PBS).
5. Loosen the cell pellet by tapping onto the tube and resuspend the cells in 100 µL of the antibody solution prepared in step 3.4. Keep the samples in the dark from this step onward.
6. Incubate the samples for 1 h at room temperature.
7. Centrifuge the cells and discard the supernatant carefully by pipetting. Take care not to aspirate the pellet.
8. Loosen the cell pellet by tapping onto the tube and resuspend the cells in 100-250 µL of DNA staining solution (1 µg/mL DAPI/PBS).
   1. Use 100 µL if 1-2 x10⁵ cells are present and increase the volume for higher cell numbers (250 µL for ≥1 x 10⁶ cells). Proceed directly with the next step or store the samples in the dark at 4 °C for up to 2 weeks.
      NOTE: For some cell types optimizing the DAPI concentration can help to improve the separation of the cell cycle phases.
9. Pipet the samples through the cell strainer cap of a sample tube with a mesh pore size of 35 µm.

4. Measurement

1. Place the samples on ice, start the flow cytometer (see the Table of Materials) configured with an optical setup according to Table 1 and press the Prime button. If required, switch on the ultraviolet laser (355 nm wavelength) separately and set the power to 20 mW using the appropriate software.
2. Open the acquisition software (see Table of Materials), log in and create a new experiment by clicking the New Experiment button on the Browser toolbar.
3. Use the Inspector window to customize the name of the experiment and choose '5 Log Decades' for plot display.
4. Click the New Specimen button in the Browser toolbar and expand the new entry by clicking the '+' symbol at its left side to show the first Tube. Select the respective icon and type to rename the Specimen (e.g., cell type) and the Tube (sample identifier). Click the Tube Pointer of the first Tube (arrow-like symbol at its left) to turn it green (active).
5. Open the Parameters tab in the Cytometer window and choose the parameters according to Table 1. Delete all unnecessary parameters.
   NOTE: the parameter names may vary depending on the custom presets (e.g., Cy3 instead of Alexa555). Make sure that the selection matches the optical filters and detectors in Table 1. The light paths of all fluorophores are fully independent in this setup and compensation of spectral overlap is not required; however, it might be necessary if another optical setup is used.
6. Open the Worksheet window and create plots according to Figure 1. Draw 2 dot plots and 4 histograms using the corresponding toolbar buttons and click the axis labels to choose the appropriate parameters (front scatter (FSC-A) versus side scatter (SSC-A), DAPI-W versus DAPI-A and a histogram for DAPI-A and each antibody-coupled fluorophore.
7. Attach a control sample to the cytometer and press the Run button on the instrument. Select the first tube in the Browser window of the software and click the Acquire Data button in the Acquisition Dashboard. Adjust the sample injection volume using the Low, Mid or High buttons and the fine tuning wheel on the instrument. Preferably work at Low setting, but try to acquire at least 100 events/second (see Acquisition Dashboard).
8. Adjust the detector voltages for FSC, SSC and DAPI in the Parameters tab of the Inspector window using the dot plots in Figure 1 as a guideline. Switch to logarithmic scale for the FSC and SSC parameters if the cell population appears too dispersed on a linear scale.
9. Press the Standby button on the cytometer and continue with the worksheet setup in the software.
   1. Use the Polygon Gate tool to define the Cells population in the FSC-A versus SSC-A plot and the Rectangle Gate tool to define the SingleCells population in the DAPI-W versus DAPI-A plot. Press Ctrl + G keys to show the Population Hierarchy and click on the default gate names to rename them.
   2. Subsequently right-click on all histograms and choose Show Populations | SingleCells from the context menu.
10. Optimize the detector voltages for the antibody-coupled fluorophores to cover the full dynamic range by subsequently acquiring control and treated samples. Maximize the signal-to-noise ratio and avoid detector saturation. Make sure that the Alexa488 peak in the SingleCells population is neither truncated in the control nor the treated sample.
11. Press the Standby button on the cytometer and optionally perform steps 4.11.1-4.11.3 in the Worksheet window of the software to get a rough estimate of treatment effects during sample acquisition.
    1. Select SingleCells in the Population Hierarchy and use the Rectangle Gate tool to define the G1 population in the DAPI-W versus DAPI-A plot. Right-click at the Alexa488 histogram and choose Show Populations | G1 from the context menu.
    2. Right-click at the Alexa488 histogram and choose Create Statistics View from the context menu. Right-click at the Statistics View and choose Edit Statistics View. Go to the Statistics tab and activate the checkbox for the median of the Alexa488 signal in the G1 population (deactivate all other options).
    3. Select SingleCells in the Population Hierarchy and use the Interval Gate tool to define the subG1, M and Casp3+ populations in the DAPI-A, Alexa555-A (Cy3-A in Figure 1) and Alexa647-A histograms.
12. Press the Run button on the cytometer and measure the samples using the Acquisition Dashboard in the software. Set the stopping gate to All events or the Cells gate (if numerous small particles are present) and the number of events to record to 10,000.
13. Click Next Tube to create a new sample, rename it in the Browser window, click Acquire Data to start the acquisition and Record Data to start recording.
14. Select File | Export | FCS files from the menu bar to export the data. Optionally select File | Export | Experiments to save the experiment as an additional zip file to enable reimporting the experiment at a later time point.
    NOTE: The setup of existing experiments can be easily reused with Edit | Duplicate without data.

5. Data Evaluation

1. Drag and drop the '.fcs' files into the sample browser of the flow cytometric analysis software (see Table of Materials). Apply the gating strategy shown in Figure 2. Make sure that the gates fit to the corresponding population in all of the samples before proceeding with the next daughter gate.
   1. To apply changes to all samples, select the changed gate in the sample browser, copy it by pressing Ctrl + C, select the parent gate, press Ctrl + Shift + E to select the equivalent nodes in all samples and press Ctrl + V to paste or overwrite the gate. Do not use group gates.
   2. Double-click on the first sample in the browser to open the SSC-A vs. FSC-A plot. Use the Polygon tool in the toolbar to define the Cells population (Figure 2, plot 1), excluding debris from the analysis. Make sure that the gate is wide enough towards the upper right corner to accommodate treatment-related shifts, but restrict the border facing to the lower left corner of the plot to reliably exclude the cell.
   3. Double-click on the Cells gate to open a new plot window and change the axes to DAPI-W (vertical) vs. DAPI-A (horizontal) by clicking on the axis labels. Use the Rectangle tool to define the SingleCells population (Figure 2, plot 2), excluding cell doublets or clumps from the analysis (doublets of G1 cells have the same DAPI-A intensity as G2 and M cells, but a considerably higher DAPI-W value).
      NOTE: Varying cell counts in different samples will cause shifts in the overall DAPI-A signal strength due to equilibrium binding of DAPI to DNA. This will not affect the cell cycle analysis, but it may be necessary to adjust the right border of the single cell gate sample by sample to account for these shifts.
   4. Double-click on the SingleCells gate to open a new plot window and change the axes to show the DAPI-A histogram. Use the Bisector tool to distinguish single cells with normal DNA content (CellCycle population) from apoptotic cells with degraded DNA (subG1 population). Subsequently select the new gates in the browser and press Ctrl + R to rename them accordingly (Figure 2, plot 3).
   5. Select the CellCycle gate in the browser and choose Tool | Biology | Cell Cycle... from the menu bar to open the cell cycle modelling tool. Choose Dean-Jett-Fox^25,26 in the Model section to estimate the frequency of cells in G1, S and G2/M phase (Figure 2, plot 4). Use constraints only in case of poor modelling performance (minimize the Root Mean Square deviation between model and data).
   6. Create gates for G1 (Ellipse tool), S (Polygon tool) and G2 + M (Ellipse tool) phase in the DAPI-W vs. DAPI-A plot of the CellCycle population to enable cell cycle-specific γH2AX measurement (Figure 2, plot 5). Do not use the modelling tool for automatic cell cycle gating based on the DAPI-A histogram, as this can be inaccurate.
      NOTE: It may be necessary to move the gates along the DAPI-A axis sample by sample to account for the aforementioned shifts in overall DAPI signal strength. Only move the three gates as a group and do not change the shape of individual gates to avoid bias.
   7. Use the Bisector tool to distinguish phospho-histone H3-positive (M) and -negative (M-) cells in the Alexa555-A histogram of the CellCycle population (Figure 2, plot 6). Hold Ctrl and select the gates G2 + M and M-. Press Ctrl + Shift + A to create the G2 (G2+M & M-) gate.
   8. Use the Bisector tool to distinguish caspase-3-positive (Casp3+) and -negative (Casp3-) cells in the Alexa647-A histogram of the SingleCells population (Figure 2, plot 7). Set the threshold such that the average of the Casp3+ population in the untreated controls amounts to ~0.8% to assure high sensitivity and minimize assay-to-assay variations.
   9. Press Ctrl + T to open the Table Editor and configure it according to Figure 3. Drag and drop the different populations from the sample browser into the Table Editor and double-click on the rows to change the statistic, parameter and name settings. Remove the unnecessary rows that are automatically added after the drag and drop of the cell cycle modelling icon by selecting the rows and pressing Del.
   10. Choose To File, the format and destination in the Output section of the menu ribbon and click Create Table to export the data as '.xlsx' file.
2. Use table calculation software (see Table of Materials) for further data analysis according to Supplementary File 1 (FACS_Analysis_Template_(1).xlsx).
   1. Correct the frequencies of cells in the different cell cycle phases such that their sum amounts to 100% by applying the formula X' = X * 100 / Σ(all cell cycle phases), with X': corrected value, X: raw value, to each cell cycle phase.
      NOTE: Deviations from a sum of 100% occur due to inaccuracies in the cell cycle modelling, but are usually small (<5%).
   2. Normalize the median γH2AX intensities to the DNA content in the different cell cycle phases by dividing the values in S phase by 1.5 and in G2 and M phase by 2.0.
   3. To calculate the combined normalized γH2AX level in the whole cell population, use the formula I_A = I_G1 * G1 + I_S * S + I_G2 * G2 + I_M * M, where I_A, I_G1, I_S, I_G2, I_M are normalized median γH2AX intensities of all, G1, S, G2, M cells respectively and G1, S, G2, M are corrected frequency of cells in the respective cell cycle phase.
   4. For the normalized γH2AX levels and the frequency of subG1 and caspase-3-positive cells, subtract the average value of the untreated controls from each sample.
   5. Calculate the mean and standard deviation of each parameter from all replicate samples and plot the results into diagrams.

Wyniki

Human U87 or LN229 glioblastoma cells were irradiated with 4 Gy of photon or carbon ion radiation. Cell cycle-specific γH2AX levels and apoptosis were measured at different time points up to 48 h after irradiation using the flow cytometric method presented here (Figure 3). In both cell lines, carbon ions induced higher γH2AX peak levels that declined slower and remained significantly elevated at 24 to 48 h compared to photon radiation at the same physical dose (

Dyskusje

The featured method is easy to use and offers a fast, accurate and reproducible measurement of the DNA damage response including double-strand break (DSB) induction and repair, cell cycle effects and apoptotic cell death. The combination of these endpoints provides a more complete picture of their interrelations than individual assays. The method can be widely applied as a comprehensive genotoxicity assay in the fields of radiation biology, therapy and protection, and more generally in oncology (e.g., for environmental r...

Materiały

Name	Company	Catalog Number	Comments
1,000 µL filter tips	Nerbe plus	07-693-8300
100-1,000 µL pipette	Eppendorf	3123000063
12 mm x 75 mm Tubes with Cell Strainer Cap, 35 µm mesh pore size	BD Falcon	352235
15 mL tubes	BD Falcon	352096
200 µL filter tips	Nerbe plus	07-662-8300
20-200 µL pipette	Eppendorf	3123000055
4’,6-Diamidin-2-phenylindol (DAPI)	Sigma-Aldrich	D9542	Dissolve in water at 200 µg/mL and store aliquots at -20 °C
Alexa Fluor 488 anti-H2A.X Phospho (Ser139) Antibody, RRID: AB_2248011	BioLegend	613406	Dilute 1:20
Alexa Fluor 647 Rabbit Anti-Active Caspase-3 Antibody, AB_1727414	BD Pharmingen	560626	Dilute 1:20
BD FACSClean solution	BD Biosciences	340345	For cytometer cleaning routine after measurement
BD FACSRinse solution	BD Biosciences	340346	For cytometer cleaning routine after measurement
Dulbecco’s Phosphate Buffered Saline (PBS)	Biochrom	L 182	Dissolve in water to 1x concentration
Dulbecco's Modified Eagle's Medium with stable glutamin	Biochrom	FG 0415	Routine cell culture material for the example cell line used in the protocol
Ethanol absolute	VWR	20821.330
Excel software	Microsoft
FBS Superior (fetal bovine serum)	Biochrom	S 0615	Routine cell culture material for the example cell line used in the protocol
FlowJo v10 software	LLC	online order
Fluoromount-G	SouthernBiotech	0100-01	Embedding medium for optional preparation of microscopic slides from stained samples
Folded cellulose filters, grade 3hw	NeoLab	11416
LSRII or LSRFortessa cytometer	BD Biosciences
MG132	Calbiochem	474787	optional drug for apoptosis positive control
Multifuge 3SR+	Heraeus
Paraformaldehyde	AppliChem	A3813	Prepare 4.5% solution fresh. Dilute in PBS by heating to 80 °C with slow stirring under the fume hood. Cover the flask with aluminium foil to prevent heat loss. Let the solution cool to room temperature and adjust the final volume. Pass the solution through a cellulose filter.
Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit mAb (Alexa Fluor® 555 Conjugate) RRID: AB_10694639	Cell Signaling Technology	#3475	Dilute 3:200
PIPETBOY acu 2	Integra Biosciences	155 016
Serological pipettes, 10 mL	Corning	4488
Serological pipettes, 25 mL	Corning	4489
Serological pipettes, 5 mL	Corning	4487
SuperKillerTRAIL (modified TNF-related apoptosis-inducing ligand)	Biomol	AG-40T-0002-C020	optional drug for apoptosis positive control
T25 cell culture flasks	Greiner bio-one	690160	Routine cell culture material for the example cell line used in the protocol
Trypsin/EDTA	PAN Biotech	P10-025500	Routine cell culture material for the example cell line used in the protocol
U87 MG glioblastoma cells	ATCC	ATCC-HTB-14	Example cell line used in the protocol