PROTOCOL
An optimized method for measurement of gamma-H2AX in blood mononuclear and cultured cells Aida Muslimovic1, Ismail Hassan Ismail2, Yue Gao1 & Ola Hammarsten1 1Department
of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Go¨teborg University, Go¨teborg SE-413 45, Sweden. 2Department of Oncology, Cross Cancer Institute, University of Alberta, 11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada. Correspondence should be addressed to O.H. (
[email protected]).
© 2008 Nature Publishing Group http://www.nature.com/natureprotocols
Published online 26 June 2008; doi:10.1038/nprot.2008.93
Phosphorylation of histone protein H2AX on serine 139 (gamma-H2AX) occurs at sites flanking DNA double-stranded breaks (DSBs) and can provide a measure of the number of DSBs within a cell. We describe a flow cytometry-based method optimized to measure gamma-H2AX in nonfixed mononuclear blood cells as well as in cultured cells, which is more sensitive and involves less steps compared with protocols involving fixed cells. This method can be used to monitor induction of gamma-H2AX in mononuclear cells from cancer patients undergoing radiotherapy and for detection of gamma-H2AX throughout the cell cycle in cultured cells. The method is based on the fact that H2AX like other histone proteins are retained in the nucleus when cells are lysed at physiological salt concentrations. Cells are therefore added without fixation to a solution containing detergent to lyse the cells along with a fluorescein isothiocyanate-labeled monoclonal gamma-H2AX antibody, DNA staining dye and blocking agents. The stained nuclei can be analyzed by flow cytometry to monitor the level of gamma-H2AX to determine the level of DSBs and DNA content and to determine the cell cycle stage. The omission of fixation simplifies staining and enhances the sensitivity. This protocol can be completed within 4–6 h.
INTRODUCTION Ionizing radiation (IR) and many chemotherapeutic agents like etoposide kill cancer cells by induction of DNA DSBs1. However, the clinical effects of DSB-inducing agent are highly variable among individuals so that the same dose of IR can result in severe side effects, whereas other patients show no obvious sign of the delivered dose. It would therefore be advantageous if we could measure the DNA damage response in each patient and use this signal to guide the dosing. Unfortunately, no method can currently directly measure DSBs at relevant doses of radiation, as only 1–10 DSBs are sufficient to induce toxicity. At higher levels of radiation, it is possible to use agarose gel-based methods (CFGE (constant field gel electrophoresis)2–4 or PFGE (pulsed field gel electrophoresis)5) to directly measure DSBs. Methods that measure the more frequent, and less toxic, single-stranded DNA breaks include the comet assay6–8, unwinding assay9 and the newly developed alkaline FAR (fraction of activity released) assay (Susanne Nystro¨m, A.M., I.H.I, Jonas Nygren and O.H., unpublished manuscript). These assays can readily measure single-stranded DNA breaks at doses well below 1 Gy of radiation, but give little information concerning the level of DSBs. Therefore, the only available option to monitor DSBs at relevant doses is to measure the cellular response to DSBs. An early event after introduction of DSBs is the phosphorylation of a special form of histone 2A, denoted as H2AX, that is part of 10% of all nucleosomes, although H2AX content can vary depending on the cell type10–12. H2AX contains a distinct C-terminal extension, with a consensus phosphorylation at serine 139. The related DNA-activated kinases ATR, ATM and DNA-PK are responsible for the formation of several thousands of phosphorylated H2AX, denoted as gamma-H2AX, in a 2-Mbp region surrounding the DSB within 15 min after its formation10,13–17. The local formation of gamma-H2AX allows microscopic detection of distinct foci by fluorescent gamma-H2AX-specific antibodies that most likely represent a single DSB18–20. The
potential to detect a single focus within the nucleus makes this the most sensitive method currently available for detecting DSBs in cells. This manual method is, however, labor intensive and will be difficult to adapt to clinical practice. In contrast, flow cytometry allows simple detection of gamma-H2AX in a large number of cells and is easy to use in a clinical setting21. Several reports show that the level of gamma-H2AX as detected by flow cytometry correlates well with the number of DNA strand breaks, to the level of cell death and radiosensitivity22–26. We have developed a simple flow cytometry method optimized for measurement of gamma-H2AX in nonfixed blood cells27. H2AX is a nucleosomal protein and therefore retained in the nucleus along with other histones when cells are lysed at physiological salt concentration. It is therefore possible to lyse cells and stain the nuclei with a gammaH2AX antibody without fixation. In fact, staining of nonfixed cells significantly improves the signal in irradiated cells and lowers the background signal in nonirradiated cells resulting in a significantly improved detection of low levels of gamma-H2AX (Fig. 1). This effect is particularly pronounced in primary mononuclear cells. All nucleated cell types in blood, including the short-lived neutrophils, induce gamma-H2AX in response to DSBs27. It is therefore possible to simply lyse the red blood cells and analyze gamma-H2AX in unsorted white blood cells. We have, however, found higher reproducibility if mononuclear cells are prepared by density centrifugation before gammaH2AX analysis. Thrombocytes that always contaminate density gradient-purified mononuclear cells do not affect the data analysis, as thrombocytes lack DNA and are lysed in the staining procedure. The omission of fixation significantly simplifies the staining procedure, as unprocessed cells can be added directly to a solution (Block-9) containing a fluorescein isothiocyanate (FITC)-labeled anti-gammaH2AX antibody to stain for gamma-H2AX and a fluorescent DNA dye to stain DNA content. The Block-9 also contains several blocking agents including small fragments of DNA that have been found to
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MATERIALS REAGENTS . Cell lines, EDTA blood samples . NaH2PO4 (Sigma-Aldrich, cat. no. S8282) . Na2HPO4 (Sigma-Aldrich, cat. no. S7907) . NaCl (Sigma-Aldrich, cat. no. S7653) . Bovine serum albumin (BSA) (Sigma-Aldrich, cat. no. A7906) . NaN3 (Sigma-Aldrich, cat. no S8032) ! CAUTION Very toxic if swallowed; contact with acids liberates very toxic gas; toxic to aquatic organisms. . EDTA, Titriplex III (Merck, cat. no. 1084180) . Triton X-100 (Bio-Rad, cat. no. 161-0407) . PFA (Sigma-Aldrich, cat. no. 158127) . TRIZMA, Tris-HCl (Sigma-Aldrich, cat. no. T5941) . NaVO3 (Sigma-Aldrich, cat. no. 590088) ! CAUTION Toxic if swallowed; irritating to eyes, respiratory system and skin. . Na2MoO4 (Sigma-Aldrich, cat. no. 243655) ! CAUTION Irritating to eyes, respiratory system and skin. . NaF (Sigma-Aldrich, cat. no. S7920) ! CAUTION Toxic if swallowed; contact with acids liberates very toxic gas; irritating to eyes and skin. . Mouse serum (Sigma-Aldrich, cat. no. M5905) . Ribonulease A (RNaseA) from bovine pancreas (Sigma-Aldrich, cat. no. R5125; see REAGENT SETUP) . Deoxyribonucleic acid sodium salt, Type XIV: from herring testes (Sigma-Aldrich, cat. no. D6898; see REAGENT SETUP) . Mouse monoclonal anti-H2AXS139ph FITC conjugate (Millipore, cat. no. 16-202A) . Lymphoprep (Axis-Shield, cat. no. 1114544) . Growth medium m CRITICAL Appropriate culture medium should be chosen depending on the cell line. . Vybrant Dye Cycle Violet Stain (Invitrogen, Molecular Probes, cat. no. V35003) . Etoposide (Sigma-Aldrich, cat. no. E1383) ! CAUTION Toxic. Harmful if swallowed, may cause cancer. . Trypsin-EDTA 10, 0.5% (wt/vol) Trypsin, 5.3 mM EDTA (Invitrogen, cat. no. 15400-054) EQUIPMENT . Eppendorf tubes (1.5 ml) . Falcon tubes (15 ml) . Fluorescence-activated cell sorting (FACS) tubes
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100 No CLM CLM Relative gamma-H2AX signal
enhance the gamma-H2AX staining. After a 3-h incubation (Fig. 2), which is done on ice to prevent phosphatase action, the nuclei can be analyzed directly in a flow cytometer using FITC channel to detect gamma-H2AX and the violet channel for DNA content. Dual staining for gamma-H2AX and DNA content allows measurement of gammaH2AX signal in different cell cycle stages and allows gating so that signal from degraded nuclei and debris can be omitted. The method specifically measures gamma-H2AX, as no signal was obtained in irradiated H2AX / cells27. The gamma-H2AX signal increases linearly in the dose range from 0.3 to 20 Gy (ref. 27), meaning that it is possible to detect DSB induction below 1 Gy of IR. As most radiotherapy regimens use 2–5 Gy radiation doses, the method can be used to measure DSB induction in patients undergoing radiotherapy (see ANTICIPATED RESULTS). This method can also be used to measure gamma-H2AX levels throughout the cell cycle in any mammalian cell line. One drawback with the method is that it is not possible to stain for cell surface markers and other phosphorylation events. For these applications, cells must be fixed before addition to Block-9, resulting in less-sensitive measurement of the gamma-H2AX signal. In addition, nuclei from some cell lines and mononuclear cells prepared from old blood samples have a tendency to aggregate in Block-9, especially if staining is performed overnight. For these cells, a variant of the protocol involving limited paraformaldehyde (PFA) fixation of the
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Figure 1 | PFA-fixation results in lower gamma-H2AX signal. Blood mononuclear cells were treated or not treated with the DSB-inducing drug, calicheamicin (CLM), for 30 min at 37 1C and fixed or not fixed with 2% PFA for 10 min before gamma-H2AX staining and analysis.
nuclei must be used (see Box 1). This fixation protocol has been developed to interfere minimally with the gamma-H2AX staining. Another potential problem with the method is that the cells cannot be frozen or stored for longer periods before analysis.
. FACSAria Flow Cytometer . Phillips RT 100 X-ray machine . CO2 incubator REAGENT SETUP PBS 1.9 mM NaH2PO4, 8.1 mM Na2HPO4, 154 mM NaCl, pH 7.2. PBS is stable at room temperature (23 1C) and can be stored for 2–3 years. Block-9 staining buffer Phosphate-buffered saline supplemented with 1 g liter 1 of BSA, 8% (vol/vol) mouse serum, 0.1 g liter 1 of RNaseA, phosphatase inhibitors (10 mM NaF, 1 mM Na2MoO4, 1 mM NaVO3), 0.25 g liter 1 of herring sperm DNA, 0.1% Triton X-100, 5 mM EDTA, 0.05% (wt/vol) NaN3, 0.1 g liter 1 of RNaseA. Block-9 staining buffer should be filtered through a 0.22-mm sterile filter after preparation and stored at 4 1C until usage. Block-9 can be stored for at least 2 weeks without significant loss of gamma-H2AX staining potential. The antibodies should be added to the Block-9 buffer on the day of the experiment (see Step 2). Suspension buffer Phosphate-buffered saline supplemented with 1 g liter 1 of BSA. 120
Relative gamma-H2AX signal
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PROTOCOL
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Figure 2 | Effect of different staining time. Human melanoma cells (G361) were treated with the DSB-inducing drug, calicheamicin (CLM 10 nM), for 30 min at 37 1C, stained in Block-9 staining solution for 0.5, 1, 2, 3, 6 and 24 h before being analyzed by flow cytometry.
© 2008 Nature Publishing Group http://www.nature.com/natureprotocols
PROTOCOL Deoxyribonucleic acid sodium salt, Type XIV: from herring testes Dissolve DNA at 10 g liter 1 in 25 mM EDTA, pH 8.2. Molecular weight of the DNA should not exceed 500 bp. m CRITICAL Addition of chromosomal DNA not larger then 500 bp results in a specific reduction of the background signal, as well as an increase of the signal in cells treated with the DSB-inducing agents27. High molecular weight DNA induces aggregation of nuclei and may result in nonreproducible results. We recommend checking the molecular weight of the DNA by running 10–20 mg of the DNA on a 1% agarose gel using standard conditions and staining with 1 mg ml 1 of ethidium bromide. If significant amount of DNA with a length above 500 bp is detected, the DNA solution should be sonicated. RNaseA When dissolved, RNaseA should be made free from DNase by boiling according to the recommendations of the manufacturer. Prepare a stock solution of 5 mg ml 1 in 10 mM sodium acetate. Heat the RNaseA solution up to 100 1C for 15 min, allow to cool down to room temperature, adjust to pH 7.4 with acetic acid and filtrate through a 0.2-mm filter. The DNase-free RNaseA solution should be stored at 70 1C and are stable for at least 1 year. m CRITICAL It is important to boil RNaseA in sodium acetate and high pH, as boiling of RNaseA at neutral or low pH will lead to precipitation. PFA-fixation buffer 25 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1 mM EDTA, 0.1 g liter 1 of BSA, phosphatase inhibitors (10 mM NaF, 1 mM Na2MoO4, 1 mM NaVO3), 0.2% PFA. Etoposide Dissolve in DMSO to a final concentration of 50 mM, store at 70 1C.
BOX 1 | PFA-FIXATION OF NUCLEI TIMING 10 min
The PFA-fixation protocol should be used for (i) mononuclear cells prepared from blood samples stored at room temperature for longer than 18 h (see Step 1 B(i)) or (ii) cell lines with fragile nuclei that form aggregates during staining in Block-9 (see Step 4). The protocol stabilizes the nuclei by limited fixation that interferes minimally with gamma-H2AX staining. ? TROUBLESHOOTING 1. Treat or irradiate and prepare 200,000 cells as described in Steps 1A(i–vii) and 1B(i–xi). 2. Resuspend the cell pellet in 50 ml of PFA-fixation buffer and vortex briefly. 3. Store on ice for 5 min. 4. Transfer 25 ml of the partially fixed nuclei to 150 ml of Block-9, incubate for 2 h and proceed with Steps 4–6.
PROCEDURE Cell isolation and preparation TIMING 1–2 h 1| Cells should be prepared using option A for tumor cell lines, option B for human mononuclear cells from blood or option C if gamma-H2AX analysis is going to be performed on blood samples from patients undergoing radiotherapy. (A) Preparation of tumor cell lines for gamma-H2AX measurements TIMING 1 h (i) For analysis of gamma-H2AX, at least 100,000 cells per sample are required. (ii) Dilute etoposide to 100 mM in 1 ml of growth medium. ? TROUBLESHOOTING (iii) Discard the medium from the adherent cells. (iv) Add an appropriate volume (1 ml) of etoposide containing medium (from Step 1A(ii)) to one well of cells. Incubate at 37 1C in a cell incubator for at least 30 min. (v) Wash cells with PBS and trypsinize by adding 1 ml of 1 Trypsin-EDTA until the cells detach from the surface or for a maximum of 5 min at 37 1C in a cell incubator. ’ PAUSE POINT If the cell detachment requires longer incubation at 37 1C than 5 min, the concentration of trypsin-EDTA should be increased. ? TROUBLESHOOTING (vi) Suspend the cells in 1 ml of cold PBS and wash once with cold PBS. Keep cells on ice. (vii) Centrifuge cells at 3,700g r.p.m. in a benchtop centrifuge for 4 min at 4 1C, and then remove supernatant. (viii) Resuspend the cell pellet in 50 ml of suspension buffer. Keep on ice. ’ PAUSE POINT All cell types that we have tested can be stored on ice in PBS or appropriate cell culturing medium for 18 h before addition to Block-9 without significant loss of gamma-H2AX signal. It is not possible to freeze cells before the staining procedure. (B) Preparation of human mononuclear cells from blood samples for gamma-H2AX measurements TIMING 2 h (i) Collect 3 ml of EDTA blood from patients no longer than a few hours before irradiation. The blood can be stored on ice or at room temperature. Brief PFA-fixation of nuclei is recommended before gamma-H2AX staining if the blood samples have been stored at room temperature for longer than 18 h (see Box 1). m CRITICAL STEP The fraction of gamma-H2AX responsive mononuclear cells decreases from 100% to 70% after storage for 18 h on ice or at room temperature. ? TROUBLESHOOTING (ii) Mix the blood well and add 1 ml to a cell culture plate with an inner diameter of 33 mm. m CRITICAL STEP The thickness of the blood layer in the plate should not be above 1 mm to make sure that the blood receives an even radiation dose. If containers of other dimensions are used, adjust the blood volume accordingly. ? TROUBLESHOOTING (iii) Irradiate the blood with the desired dose (0.3–20 Gy). We use Philips RT 100 X-ray machine with an acceleration voltage of 100 kV. Store the irradiated blood on ice. ? TROUBLESHOOTING
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PROTOCOL (iv) Pipette the irradiated blood into 1.5-ml Eppendorf tubes and incubate for 30 min in a 37 1C cell incubator or water bath. ’ PAUSE POINT It takes 15 min to fully phosphorylate H2AX surrounding a DSB at 37 1C. Primary mononuclear cells show little removal of gamma-H2AX signal after 1 h incubation at 37 1C. Therefore, incubation at 37 1C can be prolonged up to 1 h without any loss of the signal. (v) Prepare 1.5-ml Eppendorf tubes with 0.5 ml of lymphoprep solution in each tube. Store at room temperature. (vi) Prepare 15-ml Falcon tubes with 14 ml of ice-cold PBS in each tube. Keep on ice. (vii) Mix 0.5 ml of blood (from Step 1 B(iv)) with 0.5 ml of room-temperature PBS. (viii) Pipette 1 ml of blood/PBS mixture (from Step 1 B(vii)) carefully on the layer of 0.5 ml of lymphoprep solution (from Step 1 B(v)). Make sure that the solutions do not mix. ? TROUBLESHOOTING (ix) Centrifuge at 1,200g for 10 min at 15–20 1C. m CRITICAL STEP Centrifugation at 4 1C will give poor separation. Therefore, this centrifugation step should be done at 15–20 1C. (x) Pipette off all solution above the erythrocyte pellet and add this lymphoprep supernatant to the prepared Falcon tubes with 14 ml of PBS from Step 1 B(vi). Mix. m CRITICAL STEP Some of the mononuclear cells may attach to the tube wall. Make sure to scrape these cells by the pipette tip before pipetting off the supernatant. ? TROUBLESHOOTING (xi) Centrifuge the Falcon tubes at 1,200g for 10 min at 4 1C and discard the PBS. (xii) Resuspend the cell pellet in 50 ml of suspension buffer. Store on ice. (C) Collection and preparation of blood samples from patients undergoing radiation treatment for gamma-H2AX measurements TIMING 2 h (i) The following blood samples should be included: (i) before irradiation of the patient; (ii) 1 h after irradiation of the patient; and (iii) in vitro irradiated blood from the same patient. (ii) Collect 3 ml of EDTA blood from the patient before irradiation. Keep on ice. m CRITICAL STEP A portion of this sample should be irradiated with the same dose as the patients receive and used as in vitro irradiated control (see Step 1 C(ii)). (iii) Irradiate 1 ml of the EDTA blood sample taken from the patient before irradiation (in Step 1 C(ii)) as described in Steps 1 B(ii–iv). (iv) Collect a 3-ml EDTA blood sample 60 min after irradiation of the patient. Keep on ice. ’ PAUSE POINT We have found that the maximum number of ‘high gamma-H2AX cells’ (see ANTICIPATED RESULTS) are present B1 h after irradiation of the patient. (v) Prepare mononuclear cells from all three blood samples (blood taken before irradiation of the patient (from Step 1 C(ii)); in vitro irradiated blood (from Step 1 C(iii)); and blood taken after irradiation of the patient (from Step 1 C(iv)), as described in Steps 1 B(v-xi)).
Gamma-H2AX staining TIMING 3–18 h 2| Determine the total volume of Block-9 buffer required for the experiment by multiplying the number of samples by 150 ml. Add anti-H2AXS139ph FITC conjugate to the final concentration of 0.6 mg ml 1 in Block-9 staining buffer. Prepare FACS tubes with 150 ml of Block-9 buffer in each. Store on ice. 3| Add cell suspensions to the FACS tubes containing 150 ml of Block-9 buffer. The total number of cells in each FACS tube should optimally be between 50,000 and 150,000. Vortex briefly and incubate for 2 h at 0–4 1C protected from light. ? TROUBLESHOOTING 4| Add Vybrant Dye Cycle Violet Stain to a final concentration of 0.5 mM (dilute the stock solution 1:1,000), vortex and incubate for an additional 1 h at 0–4 1C protected from light. ’ PAUSE POINT Samples must be incubated with the anti-gamma-H2AX antibody for at least 3 h for optimal gamma-H2AX staining. Longer incubations only slightly increase the gamma-H2AX signal (Fig. 2). Mononuclear cells can be stained on ice overnight with minimal loss of signal quality (Fig. 2). Many tumor cell lines that we have tested show signs of nuclear aggregation after 18 h staining, resulting in less reproducible results. This problem can be prevented by the PFA-fixation of nuclei protocol (Box 1). ? TROUBLESHOOTING 5| Dilute the samples by addition of 350 ml suspension buffer and vortex briefly. 6| Analyze the samples by flow cytometry. Sample analysis can be preformed on FACSAria flow cytometer using 488 and 405 nm lasers for excitation of FITC and Vybrant Dye Cycle Violet Stain, respectively. Emission is detected with the filter/bandpass: 1190 | VOL.3 NO.7 | 2008 | NATURE PROTOCOLS
PROTOCOL 105
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Figure 3 | Gamma-H2AX in different cell cycle stages upon etoposide treatment. Human melanoma cells (G361) untreated or treated with 75 mM etoposide (which induces topoisomerase II linked DNA DSBs in cellular DNA) for 30 min at 37 1C stained for gamma-H2AX and DNA content. Sample analysis preformed on FACSAria flow cytometer using 488 and 405 nm excitation laser, respectively. Emission detected with the filter/bandpass: 450/40 for FITC and 530/30 for Vybrant Dye Cycle Violet Stain resulting in two nonoverlapping signals. The gamma-H2AX versus DNA content plot indicates that gamma-H2AX levels are similar throughout the cell-cycle phases upon etoposide treatment. Cell cycle phases G1, S and G2 are indicated in the figure.
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© 2008 Nature Publishing Group http://www.nature.com/natureprotocols
DNA content
450/40 for FITC and 530/30 for Vybrant Dye Cycle Violet Stain from which two nonoverlapping signals will be obtained. Therefore compensations are not required. Plot FITC-signal versus Vybrant Dye Cycle Violet Stain signal in a dot plot to obtain gamma-H2AX levels throughout the cell cycle (Fig. 3). ? TROUBLESHOOTING
TIMING Step 1A: 1 h Step 1B: 2 h Step 1C: 2 h Box 1: 10 min Step 2: 10 min Step 3: 2 h Step 4: 1 h Step 5: 10 min Step 6: 30 min ? TROUBLESHOOTING Troubleshooting advice can be found in Table 1. TABLE 1 | Troubleshooting table. Step 4
6
4
Problem Nuclear aggregation
No signal
Nonreproducible gamma-H2AX signal
Possible reason Too many cells
Solution Count cells before addition to Block-9
High-passage cells. Cell type with fragile nuclei
Use PFA-fixation protocol (Box 1)
Cells prepared from old blood sample
Use PFA-fixation protocol (Box 1)
Degraded antibody
Avoid freeze and thawing of the antibody or prepare aliquots of the antibody and store them at 20 1C
Flow-cytometer voltage not properly adjusted
Use a positive control (a stained cell suspension or fluorescent beads) to adjust the setting of the flow cytometer
Inappropriate flow cytometer settings
Choose appropriate filter that matches the fluorophores used in the experiment
No DSB induction (Steps 1 A(ii), 1 B(iii))
Try higher doses of etoposide or IR. Avoid frequent freeze and thawing of the drugs used, prepare aliquots of the drug and store them at 20 1C
Too long staining time results in nuclear aggregation (Steps 3 and 4)
Limit the staining time to 3 h. Make sure that the tubes are kept on ice. Use DNA signal to gate nuclear debris. Use PFA-fixation protocol (Box 1)
Aggregation of the nuclei due to the presence of high molecular weight DNA in Block-9
Check molecular weight of DNA in Block-9. Make sure not to centrifuge the stained nuclei
Cells damaged during trypsinization (Step 1 A(v))
Use lower trypsin concentration
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PROTOCOL TABLE 1 | Troubleshooting table (continued). Step
Problem
Possible reason
Solution
Uneven gamma-H2AX in Uneven radiation dose throughout the plate in vitro irradiated blood (Step 1 B(ii))
The thickness of the blood layer in the plate should not be above 1 mm
Blood stored too long before irradiation (Step 1 B(i)) Irradiate the blood within a few hours after collection Blood collected in wrong container (Step 1 B(i))
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1,4
Few cells
High signal in nontreated cells
Make sure that EDTA is used as anticoagulant
Cells lost during lymphoprep procedure (Step 1 B(viii)) Mononuclear cells attached to the tube wall should be scraped off by the pipette tip before pipetting of the supernatant above the erythrocyte pellet Nuclear aggregation (Step 4)
Limit the staining time to 3 hours or use PFA-fixation protocol (Box 1). Keep samples on ice during all times
Cells damaged during trypsinization (Step 1 A(v))
Use lower trypsin concentration
Apoptotic cells
Make sure that the cells are healthy. Wash monolayers with PBS before trypsinization to remove floating cells
Tumor cells
Many tumor cell lines and cells derived from solid tumors have a constitutive high gamma-H2AX signal
ANTICIPATED RESULTS Although this method can be used to measure gamma-H2AX in any mammalian cell type (see Table 2), we have optimized the protocol for mononuclear cells prepared from human blood. From a healthy patient, it should be expected to recover 0.5–2 106 mononuclear cells from 0.5 ml of blood. The cells recovered by this density gradient centrifugation are usually 90% T-lymphocytes, 5% B-lymphocytes and 5% monocytes, but the relative numbers of these cells may vary. All of these cell types show similar gamma-H2AX response at a given IR dose. The preparation is also contaminated with a highly variable number of thrombocytes that will not interfere with the gamma-H2AX analysis. In most cases, the cells are simply added to Block-9 that lyse the cells and stain the nuclei for gamma-H2AX and DNA content. However, for mononuclear cells prepared from blood samples stored at room temperature for over 18 h and for some cell lines (Table 2), optimal gamma-H2AX detection requires limited PFA-fixation of the nuclei before addition to Block-9 (Box 1) to prevent nuclear aggregation. Using a violet DNA dye together with gamma-H2AX antibodies, it is possible to measure gamma-H2AX levels in the cell-cycle phases and to use the DNA content to gate fragmented nuclei and other debris. We have now examined close to a hundred normal individuals and found that the gamma-H2AX signal varies below 20% in nontreated mononuclear cells. In contrast, in vitro irradiated mononuclear cells from 50 individuals show a twofold variation in the gamma-H2AX signal produced by a given dose of IR. It is therefore expected to observe a significant variation of the gamma-H2AX signal among patients who receive the same radiation dose. We and others have observed that nontreated tumor cell lines and cells recovered from solid human cancers have a significantly higher gamma-H2AX signal compared with mononuclear cells or primary cells like fibroblasts. This constitutive level of gamma-H2AX could be due to oxidative radicals produced during passage of the tumor cells. It is therefore expected that cancer cells isolated from solid tumors have a higher gamma-H2AX signal compared with primary cells. This is, however, not true for all human tumors. We have analyzed the gamma-H2AX signal in tumor cells from ten patients with hematological malignancies (acute myelogenous leukemia (AML), chronic myelogenous leukemia (KML)) and found that the gamma-H2AX signal was the same as in normal mononuclear cells. As a pilot study, we have analyzed blood samples from patients receiving 5 Gy of gamma irradiation to the pelvic area. As a control, we also analyzed the gamma-H2AX signal in in vitro irradiated mononuclear cells from the same patients (Fig. 4). Mononuclear cells from the irradiated patients show a slight increase of the gamma-H2AX signal after irradiation, corresponding
TABLE 2 | Cell types tested in the gamma-H2AX assay. Cell types Cell lines Blood cells Primary human cells aRequire
K562a Neutrophilesb Fibroblasts
PFA-fixation. bMust be prepared from fresh blood samples.
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Transf. fibroblasts T-lymphocytesa Chondrocytes
G361 Monocytes
Mo59K B-lymphocytesa
EREB
PROTOCOL
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b
Before 5 Gy 1 h after 5 Gy In vitro 5 Gy
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a Counts
Figure 4 | Gamma-H2AX measurements in patients irradiated with 5 Gy of IR to the pelvic area. (a) Mononuclear cells were prepared and stained for gamma-H2AX before irradiation (black histogram), 1 h after irradiation (red histogram) and in vitro irradiated (5 Gy) patient blood sample (blue histogram). The arrow represents ‘high gamma-H2AX cells’ that have the same signal as the in vitro irradiated control blood, indicating that a subpopulation of the patients mononuclear cells received the full 5 Gy dose. (b) The ‘high gamma-H2AX’ cell population was gated as shown by the vertical black lines in panel (a). The percentage of cells within the high gamma-H2AX gate was quantified. Percentage ‘high gamma-H2AX cells’ was plotted against the time after irradiation for three rectal cancer patients.
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Minutes after irradiation
to a dose of 0.3–0.5 Gy (Fig. 4a). The gamma-H2AX signal in these cells will likely be below the detection limit if the more conventional radiation dose of 2 Gy were used and will likely vary depending on the blood flow through the irradiated volume. However, we also observed a small fraction of mononuclear cells that apparently have received the full 5 Gy of irradiation, as their gamma-H2AX signal matched the signal observed in the in vitro irradiated mononuclear cells (Fig. 4, arrow). These mononuclear cells were likely trapped in the microcirculation in the irradiated area during the 2 min of irradiation and then released into the systemic circulation. These ‘high gamma-H2AX signal cells’ accumulate after irradiation and constitute over to 10% of all mononuclear cells 1 h after the irradiation (Fig. 4b). Apoptotic cells also express gamma-H2AX, but at 10-fold higher levels compared with the levels observed here. We therefore find it unlikely that the ‘high gamma-H2AX cells’ constitute apoptotic cells. We also found that the gamma-H2AX signal in the in vitro irradiated mononuclear cells showed little attenuation after a 2.5-h incubation at 37 1C, indicating that the gamma-H2AX signal is retained in the irradiated mononuclear cells for a significant time after the irradiation. We have analyzed three patients and found that this pattern of gamma-H2AX signal after irradiation to be identical, except for the fact that the absolute level of gamma-H2AX signal varies among the patients (Fig. 4b). The data from our pilot study need further validation but can serve as a preliminary guide on how flow cytometry-based measurement of gamma-H2AX can be used in the clinic.
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