Quantification of DNA double-strand streaks using γH2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of γH2AX foci after exposure of cells to radiation.
Cell Preparation
Irradiation
Immunofluorescence staining
Microscopy / Analysis
Figure 1. Immunofluorescence visualization of γH2AX foci (green) in untreated human keratinocytes and in cells irradiated with 2 Gy and incubated for a further 1 hour at 37°C, 5% CO2. DNA was stained with TOPRO-3 (blue). Images were acquired using a Zeiss LSM 510 Meta Confocal microscope. Bar = 10 μm.
Figure 2. Immunofluorescence visualization of γH2AX foci (green) in human keratinocytes and in cells irradiated with 2 Gy and incubated for a further 1 hour at 37°C, 5% CO2. Images were acquired using a Zeiss LSM 510 Meta Confocal microscope using 0.5 μm Z-sectioning (1-9) to ensure all foci were acquired. The images was then stacked for quantitation using Metamorph. DNA was stained with TOPRO-3 (blue). The stacked γH2AX and blue images were stacked for visualization. Bar = 10 μm.
Following exposure to ionising radiation (γ-rays), γH2AX foci form rapidly and foci numbers reach a maximum between 30-60 minutes2. Therefore, our 1 hour post-irradiation time point reflects initial DSB formation. We have used the clinically relevant radiation dose of 2 Gy for our experiment. However, the method can be used for radiation doses up to 4 Gy for detection of initial DSB formation; significant overlap of foci precludes accurate quantitation at higher doses. Higher radiation doses may be used for longer post-irradiation incubation times, as γH2AX foci are lost due to repair, resulting in quantifiable numbers. Typically, 4 hour and up to 24 hour post-irradiation incubation times are used for monitoring DNA repair. We have used the DNA stain TOPRO-3 due to limitations in the excitation lasers of our confocal microscope. More commonly, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) is used for nuclear counterstaining. Although we used a confocal microscope, epifluorescent microscopes with z-sectioning capacity are adequate. The immunofluorescence method is suitable for other adherent cancer and normal cell lines; we have tested T98G human glioblastoma and normal human endothelial cells and rat H9c2 embryonic ventricular myocytes.
Finally, quantitation of γH2AX is useful in the context of ionising radiation-induced DNA damage, for monitoring DNA damage – as illustrated by our experiment – and repair (radiation sensitivity). The assay is also useful for evaluating the efficacy of compounds that modulate cellular responses to radiation (i.e. radiation protectors and sensitizers). Further, γH2AX is emerging as a potential molecular marker in aging and disease, predominantly in cancer10.
The authors have nothing to disclose.
The support of the Australian Institute of Nuclear Science and Engineering is acknowledged. TCK was the recipient of AINSE awards. Epigenomic Medicine Lab is supported by the National Health and Medical Research Council of Australia (566559). LM is supported by Melbourne Research (University of Melbourne) and Biomedical Imaging CRC supplementary scholarships. The support of Monash Micro Imaging (Drs Stephen Cody and Iśka Carmichael) was invaluable for this work.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Keratinocyte-Serum Free Medium (K-SFM) | Media | Invitrogen | 17005042 | Keratinocyte media supplemented with human recombinant Epidermal Growth Factor 1-53 (EGF 1-53), and Bovine Pituitary Extract (BPE). |
Lab Tek lI Chamber Slides (8-well) | Chamber Slides | Nunc Denmark | NUN154534 | |
Coverslips (22x50mm) | Coverslips | Menzel-Gläser | CS2250100 | |
Bovine Serum Albumin (BSA) | Sigma-Aldrich | A7906 | BSA (1%) is used to block any non-specific antibody binding. Primary and secondary antibodies are diluted in BSA. | |
PBS (without Ca2+ and Mg2+) | Invitrogen | 17-517Q | ||
0.5% Trypsin-EDTA x10 | Invitrogen | 15400-054 | Trypsin-EDTA (0.05%) used to detach cells from culture flasks. | |
Triton X-100 | Reagent | Sigma-Aldrich | T8787 | Triton X-100 (0.1%) used to permeabilise cells. |
Paraformaldehyde | Reagent | Sigma-Aldrich | 158127 | Paraformaldehyde (4%) used to fix cells. |
Mouse monoclonal anti-phospho histone-H2AX antibody | Primary Antibody | Millipore | 16193 | Dilution of primary antibody (1:500), in 1% BSA. |
Alexa Fluor 488 goat anti-mouse IgG (H+L) | Secondary Antibody | Invitrogen | 11029 | Dilution of secondary antibody (1:500), in 1% BSA. |
TOPRO3 | DNA Stain | Invitrogen | T3605 | DNA stain commonly used: 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Can only be used with microscopes with the appropriate excitation laser. |
ProLong Gold | Anti-fade solution | Invitrogen | P36930 | Refractive index of 1.42 at 20°C. |
Tissue Culture Flask, Vented Cap | Culture Flask | BD Falcon | 353112 | |
Tissue Culture Dish (150x25mm) | Petridish | BD Falcon | 353025 | |
Coplin Jar, glass | Grale Scientific P/L | 1771-OG | ||
Staining Trough | Grale Scientific P/L | V1991.99 | ||
Gammacell 1000 Elite Irradiator | Gamma Irradiator | Nordion International Inc. | ||
Zeiss LSM 510 Meta Confocal | Confocal Microscope | |||
Metamorph | Software for Imaging analysis | Molecular Devices, USA |