1Epigenetics in Human Health and Disease, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 2Epigenomic Medicine, BakerIDI Heart and Diabetes Institute, The Alfred Medical Research and Education Precinct, 3Department of Pathology, University of Melbourne
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Vasireddy, R. S., Tang, M. M., Mah, L., Georgiadis, G. T., El-Osta, A., Karagiannis, T. C. Evaluation of the Spatial Distribution of γH2AX following Ionizing Radiation. J. Vis. Exp. (42), e2203, doi:10.3791/2203 (2010).
An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as γH2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of γH2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of γH2AX as molecular marker of DSBs, a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of γH2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between γH2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of γH2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.
Cells can be counted either by an automated method (e.g. Sysmex or Coulter counter with filter size between 5 to 20 microns) or by using the trypan blue exclusion method and a haemocytometer. Excess number of cells (>800 cells/cm2) may lead to non-uniform staining.
Irradiation and immunofluorescence staining
When assembling the cytospin apparatus ensure that the hole in filter card coincides with the funnel.
To prevent the formation of foci during the irradiation, keep the cells on ice for 5 to 10 minutes prior to and during irradiation.
Following irradiation incubate the cells at 37°C, 5% CO2 for the required time (For peak γH2AX levels 30 minutes to 1 hour; 1 hour in this example).
Do not let cells dry completely as cellular morphology may disintegrate.
In our experience, fixation with paraformaldehyde is superior to fixing with ethanol or methanol. Suspension cells require fixation for 5 minutes at RT whereas longer fixation times (15 to 20 minutes) are needed for optimal fixation of adherent cells.
We find that staining immediately yields better results than storing the fixed slides at -20°C which typically results in high background fluorescence.
Compared to Tween-80 we typically observe better signals in cells permeabilized with Triton-X 100. Depending on cell type it may be necessary to change the permeabilisation time. For example, in K562 cells permeabilization for 5 minutes at RT is sufficient whereas in adherent cells a 15 minute permeabilization is required.
We compared the blocking efficiency using serum (derived from same species as the secondary antibodies) and bovine serum albumin in PBS. BSA was more efficient in minimizing non-specific binding of secondary antibodies compared to serum. We compared different blocking times, overnight blocking at 4°C compared to 3 x 10 minute and 3 x 20 minute blocking steps at room temperature, and determined that blocking for 3 x 10 minutes was optimal.
Incubation with primary antibody at RT for 60 minutes results in reduced background staining compared to an overnight incubation at 4°C.
All incubations are performed in a humidified staining trough.
To stain for different markers on the same slide use secondary antibodies with different species specificity (according to the primary antibody) and fluorescence emission wavelength (e.g. anti-mouse Alexa 488 (green) and anti-rabbit Alexa-546 (red) in this example).
It is recommended to incubate cells in a dark moist chamber to avoid fading and drying of the secondary antibody.
It is recommended not to dry cells completely before adding anti-fade solution.
It is recommended to use confocal specific coverslips #1.5 (0.16-19 microns thick) and clear nail polish.
While placing the coverslip on slide, care should be taken to avoid the formation of air bubbles.
Images acquired using a confocal microscope reveal better spatial resolution.
Since the size of γH2AX foci may be lower than 0.5 μicrons, it is recommended to acquire images with at least a 0.5 μicron step size along the Z-axis.
A 63 x oil immersion objective lens is preferred compared to either higher or lower magnification. For imaging many cells for foci counting it is preferable to use a scan speed of 8 and image size of 1024 x 1024 pixels. To illustrate the spatial relationship between different nuclear proteins it is recommended to use a scan speed of 6 with image size of 2048 x 2048 pixels.
When imaging from multiple channels a line scan acquisition is recommended compared to a frame scan. This avoids bleaching and better resolution is achieved. Sequential scanning is used to prevent bleed through between channels.
Image analysis and foci counting can be performed using various software packages (inlcuding Metamorph, Image-J and Imaris). The procedure for foci counting using Metamorph is described here.
Care should be taken when choosing Top-Hat values. Optimal values can be obtained by visual comparison of images before and after applying filters.
It is typically the threshold values that affect foci number. Therefore, much attention is required when selecting threshold values. This can be best achieved by the counting foci number using different threshold values in cells exposed to lower than 2 Gy of γ-radiation and then compare foci numbers with manual counting (by eye). The optimal threshold value is the one that minimizes the inclusion of background and maximizes inclusion of foci.
3D reconstruction of images
To create a 3D image from a stack using Metamorph:
It is preferable to use the user specified Z-distance (optimal is 0.5 μicrons).
Line scan analysis
For line scan analysis using Metamorph:
To elucidate the spatial relationship of various chromatin markers, for example, γH2AX foci and histone methylation, it is preferable to draw lines which span regions that are both dense and poor in these markers.
For the purpose of this demonstration we used antibodies to γH2AX foci and to H3K4me, to evaluate the spatial distribution of DSBs to an epigenetic determinant of actively transcribing euchromatin. As shown in figure 1, following irradiation with 2Gy, γH2AX foci formed predominantly in regions that were dull for both H3K4me staining and for the DNA stain TOPRO-3 (brightly stained DNA regions are indicative of heterochromatin).
Figure 1. γH2AX foci form predominantly in euchromatin in response to ionizing radiation. (A) Immunofluorescence visualization of γH2AX foci (green) in human erythroleukemic K562 cells, 1 hour after γ-irradiation (2 Gy). γH2AX foci are shown in relation to H3K4me, representing actively transcribing euchromatin (red). DNA is labelled with TOPRO-3 (blue). The merged image (blue, red and green) demonstrates the exclusion γH2AX foci from heterochromatic regions. The line scan analysis (fluorescence intensity vs distance) indicates the relative distribution of the markers in a single plane. (B) Slices at different planes from the 3D reconstruction of the merged image described above.
No conflicts of interest declared.
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). This work is funded by the CRC for Biomedical Imaging Development Ltd, established and supported under the Australian Government s Cooperative Research Centres (CRC) program. 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.
|Roswell Park Memorial Institute -1640 (RPMI-1640)||Growth medium||Invitrogen||22400071||RPMI-1640, pH 7.4 medium supplemented with 10% (v/v) fetal bovine, 2mM L-glutamine, 20μg/ml gentamicin, 20mM (HEPES) N-2-Hydroxyethylpiperazine-N’-2-Ethanesulfonic Acid|
|Fetal Bovine Serum (FBS)||Sigma-Aldrich||F2442|
|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|
|Trypan blue||Sigma-Aldrich||T6146||Used to distinguish between live and dead cells.|
|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||EMD Millipore||16193||Dilution of primary antibody (1:500), in 1% BSA.|
|HistoneH3 (Mono-methyl K4)||Primary Antibody||Abcam||AB8895|
|Alexa Fluor 488 goat anti-mouse IgG (H+L)||Secondary Antibody||Invitrogen||11029||Dilution of secondary antibody (1:500), in 1% BSA.|
|Alexa Fluor 546 goat anti-rabbit IgG (H+L)||Secondary Antibody||Invitrogen||11035|
|TOPRO3||DNA Stain||Invitrogen||T3605||TOPRO3 is a DNA dye with an Abs/Em of 642/661 nm. DAPI could be used if the confocal microscope is equipped with a 405 nm laser.|
|ProLong Gold||Anti-fade solution||Invitrogen||P36930||This glycerol based mounting medium must be used with an oil based lense that matches its refractive index.|
|Tissue Culture Flask, Vented Cap||Culture Flask||BD Biosciences||353112|
|Shandon Cytospin 4||Thermo Fisher Scientific, Inc.|
|Filter Cards||Shandon, Inc.||353025|
|Coplin Jar, glass||Grale Scientific P/L||1771-OG|
|Staining Trough||Grale Scientific P/L||V1991.99|
|PAP Pen||Zymed Laboratories, Inc.||008877|
|Gammacell 1000 Elite Irradiator||Gamma Irradiator||Nordion International Inc.|
|Zeiss LSM 510 Meta Confocal||Confocal Microscope||Equipped with 3 lasers: 488 nm, 543 nm and 633 nm.|
|Metamorph||Software for Imaging analysis||Molecular Devices|