The authors would like to acknowledge the contribution of our colleagues Sandrine Roch-Lefevre and Eric Gregoire of the Institute of Radioprotection and Nuclear Safety (Paris, France). This work was supported by the Government of Canada Science and Technology program at Canadian Nuclear Laboratories (Chalk River, Ontario, Canada), the CANDU Owners Group (Toronto, Ontario, Canada), the Canadian Nuclear Safety Commission and by the Institute of Radioprotection and Nuclear Safety (Paris, France).

All mouse handling and treatment procedures should adhere to rules set forth by the legislature and/or animal care programs and approved by a local animal care committee. All methods described in this protocol were performed in accordance with the guidelines of the Canadian Council on Animal Care with the approval of the local animal care committee.
CAUTION: All work with radioactivity (including, but not limited to handling of tritium, external &#947;-irradiation, handling radioactive animal tissues, bedding waste) should adhere to rules set forth by the legislature and/or radiation protection authorities and be performed by authorized personnel in a certified laboratory and/or facility.
1. Animal Work
<ol>
	<li>Animal Grouping
	<ol>
		<li>If mice are arriving from an external organization, acclimatize animals for at least 10 days in the animal facility. If the study includes multiple treatment groups, ensure that the animal supplier can send all animals as a single batch.</li>
		<li>Randomly allocate mice to treatment groups of 10 mice per group. If using male mice of the C57BL/6 strain, have the animal supplier pre-arrange randomization of very young C57BL/6 males in their facility to mitigate aggression issues.</li>
	</ol>
	</li>
	<li>Chronic Exposure of Mice to Internal &#946;-Radiation
	<ol>
		<li>Prepare working concentrations of tritium (10 kBq/L and 1 MBq/L) in mouse drinking water by diluting the original stock of HTO and OBT (a mixture of tritiated proline, alanine and glycine amino acids) solutions.</li>
		<li>Validate the final concentrations of tritium in the drinking water by measuring radioactivity using a liquid scintillation counter. Adjust concentrations, if required.</li>
		<li>Treat animals for one month by providing access ad libitum to the tritium water. If treatment water gets low during the two week period, add more water. Otherwise, change bottles at two weeks.</li>
		<li>Treat sham-irradiated control mice identically. Keep them in a separate &#8220;clean&#8221; room or on a separate &#8220;clean&#8221; rack under positive air pressure to avoid tritium cross-contamination from the air.</li>
	</ol>
	</li>
	<li>Chronic Exposure of Mice to External &#947;-Radiation 
	Note: &#947;-irradiation facility/equipment will vary depending on the location.</li>
	<li>Using available methods of dosimetry, determine the distances from a &#947;-radiation source that have radiation fields producing the desired dose rates. Ensure homogeneity of the radiation fields within the areas covering the animal cages. 
	Note: 1.7 &#956;Gy/h is equivalent to a dose rate produced by 1 MBq/L tritium in the body.</li>
	<li>Expose mice by placing mouse cages at the distances determined in step 1.3.1 for one month. Minimize daily interruption of &#947;-irradiation to 30 min. Use thermoluminescence dosimeters to measure total absorbed doses.</li>
</ol>
2. Sacrifices and Sampling
<ol>
	<li>Prepare sterile surgical instruments, 60 mm petri dishes with inserted 70-micron cell strainers, 15 ml and 1.5 ml tubes, RPMI media supplemented with 5% fetal bovine serum (FBS) and place all within a biological safety cabinet. Ensuring sterility of the work is crucial.</li>
	<li>Dispense 5 ml of RPMI media into each 60 mm petri dish.</li>
	<li>Sacrifice mice using a method approved by the local animal care committee. Cervical dislocation is a good choice for the splenocyte work described in this protocol. However, if blood samples for other assays (e.g., for the mFISH, the cytochalasin block micronucleus or other common genetoxicity assays) are required, use intra-cardiac puncture after anaesthesia with isoflurane.</li>
	<li>Place the animal in dorsal recumbency, with the head pointing away from you. Spray the mouse down with 70% ethanol. Using forceps, grab the skin anteriorly to the urethral opening and make a small incision with scissors in the perineal region.
	<ol>
		<li>From this incision, cut along the ventral midline to the chest cavity, being careful to only cut the skin and not the muscle wall underneath. Dissect the skin away from the midline.</li>
		<li>Grasp the abdominal wall with forceps and cut along the median axis of the muscular wall to open up the abdominal cavity.</li>
	</ol>
	</li>
	<li>Excise the spleen by lightly gripping it with sterile forceps and gently pulling on it while simultaneously cutting away the connective tissue with scissors.</li>
	<li>Cut a small piece of the spleen (approximately 1/10 of the spleen) and place in a 1.5 ml tube. Snap freeze in liquid nitrogen for subsequent storage at -80 &#176;C for supplementary assays.</li>
	<li>Place the remainder of the spleen into a cell strainer inside a 60 mm petri dish with 5 ml of pre-dispensed RPMI media.</li>
	<li>Proceed to the next mouse. 
	Note: Extracted spleens can be kept in 60 mm dishes with media (for up to 2 hrs) while the rest of the mice are sacrificed. Depending on the number of people participating, between 5 and 15 mice can be processed in one day.</li>
</ol>
3. Preparation of Splenocyte Cultures
<ol>
	<li>Homogenize the spleen by mincing inside a cell strainer with sterile forceps with a curved end.</li>
	<li>Remove the cell strainer and collect the filtered cell suspension from the 60 mm dish into a 15 ml tube.</li>
	<li>Rinse the cell strainer with 5 ml of media to collect the remainder of the cells.</li>
	<li>Centrifuge tubes at 300 x g for 5 min at RT.</li>
	<li>Decant supernatant and re-suspend pellet in 10 ml of RPMI.</li>
	<li>Centrifuge tubes at 300 x g for 5 min at RT.</li>
	<li>Decant supernatant and re-suspend pellet in 5 ml of RPMI.</li>
	<li>Transfer 4 ml of cell suspension into a 25 ml tissue culture flask and remove to a CO2 incubator (37 &#176;C, 5% CO2, 80% humidity)</li>
</ol>
4. Challenging Irradiation and Fixing
<ol>
	<li>Transfer the remaining 1 ml of cell suspension from step 3.8 into a 1.5 ml tube on ice and centrifuge at 300 x g for 5 min at 4 &#176;C. DNA damage measured in this sample will represent both treatment-induced damage and t = 0 data-point for the construction of a DNA repair curve.</li>
	<li>Carefully aspirate the supernatant using a vacuum pump. Gently re-suspend pellet in 1 ml of TBS buffer (20 mM Tris pH 7.4, 150 mM NaCl, 2.7 mM KCl).</li>
	<li>Centrifuge tubes at 300 x g for 5 min at 4 &#176;C. Carefully aspirate the supernatant using a vacuum pump. Gently re-suspend pellet in 300 &#956;l of TBS.</li>
	<li>While mixing on a vortex at low speed, add 700 &#956;l of -20 &#176;C 100% ethanol. Close the lid and invert a few times to mix.</li>
	<li>Store the samples at -20 &#176;C. Splenocytes fixed in ethanol can be stored at -20 &#176;C for at least 12 months without any noticeable loss in the &#947;H2AX signal.</li>
	<li>Irradiate the splenocyte cultures from step 3.8 with a challenging &#947;-radiation dose of 2 Gy at a dose rate &#8805; 200 mGy/min using available irradiation device. 
	Note: The purpose of this irradiation is to induce DNA DSB to challenge the repair machinery. X-rays can also be used for this purpose.</li>
	<li>Immediately return the cultures to a CO2 incubator.</li>
	<li>1 hr after the challenging 2 Gy irradiation, remove the cultures from the incubator to a biological safety cabinet. To collect a representative sample aliquot, gently re-suspend cells using a pipettor and transfer 1 ml of the cell suspension to a 1.5 ml tube on ice. Return the cell cultures to a CO2 incubator.</li>
	<li>Centrifuge tubes at 300 x g for 5 min at 4 &#176;C. Carefully aspirate the supernatant using a vacuum pump. Gently re-suspend pellet in 1 ml of TBS.</li>
	<li>Repeat steps 4.3 - 4.5.</li>
	<li>24 hrs after the challenging 2 Gy &#947;-irradiation, harvest and fix a t = 24 hrs sample as described in step 4.8 &#8211; 4.10.</li>
</ol>
5. Immunofluorescent Labeling with Anti-&#947;H2AX Antibody
Note: Typically, 15 - 30 samples per day can be immunofluorescently labeled and assayed by flow cytometry in a single run. To ensure an accurate comparison between treatment groups, include sample(s) from each group. See also Reference 16&#160;for further details.
<ol>
	<li>Prepare a labeled set of tubes for the number of samples to be processed and place on ice. Use 12 x 75 mm uncapped glass tubes. Sterility is not required for this work. Include one additional historical control sample for labeling with secondary antibody only (&#8220;2nd&#160;Ab only&#8221;). Use this historical control sample in every flow cytometry run within a study.</li>
	<li>Add 0.5 ml of ice-cold TBS to each tube.</li>
	<li>Remove fixed splenocytes from -20 &#176;C, vortex for 5 sec and transfer a 0.5 ml aliquot to prepared tubes with TBS. Place the remainder of samples back into -20 &#176;C storage and keep as a backup sample.</li>
	<li>Centrifuge splenocytes at 300 x g for 5 min at 4 &#176;C. Decant supernatant and gently re-suspend pellet in 1 ml of ice-cold TBS containing 1% FBS</li>
	<li>Centrifuge cells at 300 x g for 5 min at 4 &#176;C. Decant the supernatant and gently re-suspend pellet in 1 ml of TST buffer (0.05% Triton X-100, 2% FBS in TBS). Incubate for 20 min on ice.</li>
	<li>Centrifuge cells at 300 x g for 5 min at 4 &#176;C. Decant the supernatant and gently re-suspend pellet in 200 &#956;l of primary anti-&#947;H2AX antibody diluted 1:150 in TST buffer. Use 200 &#956;l of TST for the &#8220;2nd&#160;Ab only&#8221; control sample.</li>
	<li>Position tubes on a rotating shaker at an angle of 45 - 60&#176; and shake for 1.5 h at 300 x g at RT.</li>
	<li>While tubes are incubating, prepare a sufficient volume (200 &#956;l/sample) of the goat anti-mouse secondary antibody conjugated with Alexa-488 at a 1:200 dilution in TST buffer. 
	Note: All work involving Alexa-488 conjugated antibody (steps 5.10 to 5.16 below) should be done under dim light conditions and labeled samples should be protected from light by wrapping tubes in aluminum foil.</li>
	<li>Once the 1.5 hrs incubation is completed, add 1 ml of ice-cold TBS containing 2% FBS to each tube and centrifuge at 300 x g for 5 min at 4 &#176;C. Decant the supernatant and gently re-suspend pellet in 1 ml of TBS containing 2% FBS.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C. Decant supernatant and gently re-suspend pellet in 200 &#956;l of the secondary antibody solution from step 5.8.</li>
	<li>Incubate the samples on a shaking platform for 1 hr as in step 5.7.</li>
	<li>Add 1 ml of ice-cold TBS containing 1% FBS.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C. Decant supernatant and gently re-suspend pellet in 1 ml of ice-cold TBS.</li>
	<li>Centrifuge at 300 x g for 5 min at 4 &#176;C. Decant the supernatant and gently re-suspend pellet in 0.5 ml of TBS containing 50 &#956;g/ml propidium iodide.</li>
	<li>Incubate for 5 min at RT protected from light.</li>
	<li>Analyze samples on a flow cytometer using instrument-specific protocols16. Read at least 10,000 cells per sample.</li>
</ol>

<ol>
	<li>Mitchel, R. E., Jackson, J. S., McCann, R. A., Boreham, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adaptive+response+modifies+latency+for+radiation-induced+myeloid+leukemia+in+CBA/H+mice.">The adaptive response modifies latency for radiation-induced myeloid leukemia in CBA/H mice.</a> Radiat Res. 152 (3), 273-279 (1999).</li><li>Shadley, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+adaptive+response+in+human+lymphocytes.">Chromosomal adaptive response in human lymphocytes.</a> Radiat Res. 138 (Suppl 1), S9-12 (1994).</li><li>Wiencke, J. K., Afzal, V., Olivieri, G., Wolff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+the+[3H]thymidine-induced+adaptive+response+of+human+lymphocytes+to+subsequent+doses+of+X-rays+involves+the+induction+of+a+chromosomal+repair+mechanism).">Evidence that the [3H]thymidine-induced adaptive response of human lymphocytes to subsequent doses of X-rays involves the induction of a chromosomal repair mechanism).</a> Mutagenesis. 1 (5), 375-380 (1986).</li><li>Mitchel, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dose+window+for+radiation-induced+protective+adaptive+responses.">The dose window for radiation-induced protective adaptive responses.</a> Dose-Response. 8 (2), 192-208 (2010).</li><li>Tubiana, M., Feinendegen, L. E., Yang, C., Kaminski, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+linear+no-threshold+relationship+is+inconsistent+with+radiation+biologic+and+experimental+data.">The linear no-threshold relationship is inconsistent with radiation biologic and experimental data.</a> Radiology. 251 (1), 13-22 (2009).</li><li>Gent, D. C., Hoeijmakers, J. H., Kanaar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+stability+and+the+DNA+double-stranded+break+connection.">Chromosomal stability and the DNA double-stranded break connection.</a> Nat Rev Genet. 2 (3), 196-206 (2001).</li><li>Jackson, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+and+repairing+DNA+double-strand+breaks.">Sensing and repairing DNA double-strand breaks.</a> Carcinogenesis. 23 (5), 687-696 (2002).</li><li>Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+double-stranded+breaks+induce+histone+H2AX+phosphorylation+on+serine+139.">DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139.</a> J Biol Chem. 273 (10), 5858-5868 (1998).</li><li>Schultz, L. B., Chehab, N. H., Malikzay, A., Halazonetis, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+binding+protein+1+(53BP1)+is+an+early+participant+in+the+cellular+response+to+DNA+double-strand+breaks.">p53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks.</a> J Cell Biol. 151 (7), 1381-1390 (2000).</li><li>Kinner, A., Wu, W., Staudt, C., Iliakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-H2AX+in+recognition+and+signaling+of+DNA+double-strand+breaks+in+the+context+of+chromatin.">Gamma-H2AX in recognition and signaling of DNA double-strand breaks in the context of chromatin.</a> Nucleic Acids Res. 36 (17), 5678-5694 (2008).</li><li>Sedelnikova, O. A., Rogakou, E. P., Panyutin, I. G., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+detection+of+(125)IdU-induced+DNA+double-strand+breaks+with+gamma-H2AX+antibody.">Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody.</a> Radiat Res. 158 (4), 486-492 (2002).</li><li>Yuan, J., Adamski, R., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focus+on+histone+variant+H2AX:+to+be+or+not+to+be.">Focus on histone variant H2AX: to be or not to be.</a> FEBS Lett. 584 (17), 3717-3724 (2010).</li><li>Taneja, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H2AX+phosphorylation+as+a+predictor+of+radiosensitivity+and+target+for+radiotherapy.">Histone H2AX phosphorylation as a predictor of radiosensitivity and target for radiotherapy.</a> J Biol Chem. 279 (3), 2273-2280 (2004).</li><li>Olive, P. L., Banath, J. P., Sinnott, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylated+histone+H2AX+in+spheroids,+tumors,+and+tissues+of+mice+exposed+to+etoposide+and+3-amino-1,2,4-benzotriazine-1,3-dioxide.">Phosphorylated histone H2AX in spheroids, tumors, and tissues of mice exposed to etoposide and 3-amino-1,2,4-benzotriazine-1,3-dioxide.</a> Cancer Res. 64 (15), 5363-5369 (2004).</li><li>Banath, J. P., Klokov, D., MacPhail, S. H., Banuelos, C. A., Olive, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Residual+gammaH2AX+foci+as+an+indication+of+lethal+DNA+lesions.">Residual gammaH2AX foci as an indication of lethal DNA lesions.</a> BMC Cancer. 10, 4 (2010).</li><li>Blimkie, M. S., Fung, L. C., Petoukhov, E. S., Girard, C., Klokov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+DNA+double-strand+breaks+is+not+modulated+by+low-dose+gamma+radiation+in+C57BL/6J+mice.">Repair of DNA double-strand breaks is not modulated by low-dose gamma radiation in C57BL/6J mice.</a> Radiat Res. 1815 (5), 548-559 (2014).</li><li>Runge, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+interpretation+of+ionizing+radiation-induced+gammaH2AX+foci+by+the+novel+pattern+recognition+system+AKLIDES(R).">Fully automated interpretation of ionizing radiation-induced gammaH2AX foci by the novel pattern recognition system AKLIDES(R).</a> Int J Radiat Biol. 88 (5), 439-447 (2012).</li><li>Jucha, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FociCounter:+A+freely+available+PC+programme+for+quantitative+and+qualitative+analysis+of+gamma-H2AX+foci.">FociCounter: A freely available PC programme for quantitative and qualitative analysis of gamma-H2AX foci.</a> Mutat Res. 696 (1), 16-20 (2010).</li><li>Garty, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RABIT:+a+rapid+automated+biodosimetry+tool+for+radiological+triage.">The RABIT: a rapid automated biodosimetry tool for radiological triage.</a> Health Phys. 98 (2), 209-217 (2010).</li><li>Kovalchuk, I. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+changes+in+DNA+repair+in+radiation-exposed+mice.">Age-dependent changes in DNA repair in radiation-exposed mice.</a> Radiat Res. 182 (6), 683-694 (2014).</li><li>Rube, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+in+the+context+of+chromatin:+new+molecular+insights+by+the+nanoscale+detection+of+DNA+repair+complexes+using+transmission+electron+microscopy.">DNA repair in the context of chromatin: new molecular insights by the nanoscale detection of DNA repair complexes using transmission electron microscopy.</a> DNA Repair. 10 (4), 427-437 (2011).</li><li>Amundson, S. A., Do, K. T., Fornace, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+stress+genes+by+low+doses+of+gamma+rays.">Induction of stress genes by low doses of gamma rays.</a> Radiat Res. 152 (3), 225-231 (1999).</li></ol>

The authors declare that there are no known competing financial interests.

The protocol presented in this paper is useful for conducting large scale mouse in vivo studies examining the genotoxic effects of low levels of various chemical and physical agents, including ionizing radiation. Our unique specific pathogen-free animal facility equipped with a GammaBeam 150 irradiator and a 30 meter long irradiation hall allows conducting life-long studies involving low or very low dose rate irradiations on hundreds or even thousands of mice. Smaller irradiation facilities for chronic low dose irradiation can be set up in a university or hospital environment where radiobiology studies are conducted routinely and radiation protection guidelines/regulations are implemented. However, stricter regulations normally apply to facilities designed for internal animal irradiation where handling of radionuclides, such as tritium, is carried out. Although rare, laboratories certified for such internal radiation exposure studies exist in various research institutions world-wide and their unique capabilities can be used for studies described in this protocol.
Within the protocol, we use the immunofluorescent labeling of&#160;&#947;H2AX to accurately evaluate DNA DSB rates produced by experimental exposures. More importantly, the introduction of a challenging irradiation delivered ex vivo to extracted splenocytes makes it possible to evaluate the intactness of the DNA DSB response (&#947;H2AX level at 1 hr after the challenge) and of DNA DSB repair (&#947;H2AX level at 24 hr compared to 0 hr). It should be noted that quantification of &#947;H2AX foci per cell by fluorescence microscopy may provide higher sensitivity compared to flow cytometry. For example, doses as low as 0.001 Gy were reported to produce significant increases in the number of &#947;H2AX foci per cell. However, the use of flow cytometry-based detection of &#947;H2AX provides advantages over fluorescence microscopy analysis because it requires less time for measurements, it does not require highly specialized/trained personnel and it avoids subjective decision-making related to &#947;H2AX foci recognition and counting. These advantages are critical for large scale animal studies. A typical animal experiment with 10 treatment groups and 10 mice per group will result in 100 x 3 = 300 spleen samples, if the method suggested (Figure 1) is followed. To generate data from this number of samples, approximately 100 hrs will be required using flow cytometry. Alternatively, if done by manual microscopic analyses, the same task would take at least 300 hrs, using a conservative estimate of 40 min of highly demanding microscope time per sample. Although attempts to automate analysis and scoring of &#947;H2AX foci have been made17-19, they all have a number of limitations. In particular, the round shape and small size of mouse splenocytes and lymphocytes makes it impractical to resolve all individual &#947;H2AX foci microscopically. While adherent cells have been successfully scored by automated microscopy systems for &#947;H2AX foci, no success has been demonstrated for either mouse splenocytes or lymphocytes.
The best way to eliminate day-to-day variability in the measured values of &#947;H2AX signal by flow cytometry is to include experimental group and control samples in the same run and to calculate relative &#947;H2AX changes within every run. Additional recommendations include: a) a historical control consisting of untreated and 2 Gy irradiated sample is used in every run; b) one person performs all the antibody labeling procedures; c) a single stock of buffers and a single batch of antibody, both primary and secondary, is used for the entire set of samples within one experiment. It is also vital to perform a flow cytometer quality control test recommended by the instrument manufacturer routinely and prior to each run of samples. This is required to verify the instrument&#8217;s optical alignment and fluidics system and also helps minimizing day-to-day variation in measured &#947;H2AX fluorescence between samples to be compared or pooled in one group.
It should be pointed out that the use of a larger number of time-points (e.g., 1, 2, 4, 6 hrs) post challenging irradiation may help analyze and measure short term DNA DSB repair kinetics in greater detail16. However, overall repair potential and the likeliness of delayed health effects would be represented by the residual damage measured only at 24 hrs and compared to the one observed at the initial time point (t = 0)13-15 (Figure 4).
Since basal &#947;H2AX levels are known to increase in old animals or senescent cells 20, for more accurate interpretation of the results obtained in long-term exposure mouse studies (&#62; 6 months) using the described method, the use of a young untreated group, in addition to an age-matched control, would be advisable. Although some reports indicate that &#947;H2AX may increase in proliferating cells, which would not be related to DNA DSB, this can easily be controlled by measuring S-phase and G2/M-phase cell fractions using DNA histograms (Figure 2A,&#160;D). If certain experimental conditions still prevent a researcher from using &#947;H2AX as a marker of DNA damage (due to the reasons outlined above or their combination), another protein forming DNA damage foci, such as 53BP1, can be used 21.
Although we have not attempted using this method for non-lymphoid tissues, such as muscle, heart, brain and others, it would seem difficult to process them for flow cytometry. Tissue sections and microscopy for &#947;H2AX foci counting would be the method of choice, if analysis of such tissues is required. However, other lymphoid cells, such as peripheral blood lymphocytes, bone marrow lymphocytes or thymocytes, can be used for measuring &#947;H2AX levels by the described method. The advantage of using spleen lymphocytes is the relative simplicity of cell suspension preparation and the large number of cells that can be isolated from a spleen. This becomes even more advantageous if additional end-points are included in the study. In our experience, a typical yield of splenocytes per mouse allows for the collection of at least 10 aliquots, each suitable for any of the following assays: flow cytometry, RT-qPCR for gene or miRNA expression, western blot, genomic DNA for CpG methylation assay. Thus, additional splenocyte aliquots collected in parallel to those for the &#947;H2AX end-point may be used to measure the expression of genes involved in DNA damage signaling and repair responses. Such genes as GADD45A and CDKN1A, that are normally transcriptionally up-regulated in response to cytotoxic treatments22, may provide a clue as to whether cells are properly responding to the challenging stress influence. Similarly, protein levels and post-translational modifications involved in stress responses may be assessed. Finally, it is not expected that any of the steps of the extraction procedure, if performed as described here, can induce &#947;H2AX in splenocytes.
It would be beneficial and highly recommended to sample additional organs and tissues during sacrifices to supplement DNA damage and repair data obtained for splenocytes as per presented protocol with other end-points. For example, we routinely collect bone marrow cells for the in vivo bone marrow micronucleus assay, various tissues for senescence-associated &#946;-galactosidase and DNA repair gene expression measurements. The ability to collect other tissues would depend on the available technical support during sacrifices; however, it can significantly help in the proper interpretation of results and the construction of a much more biologically relevant systemic picture of the response to the low level treatment being investigated.

Significant controversy over the potential harmful effects of either low or very low doses of ionizing radiation and public&#8217;s fear of radiation, driven by images of the Hiroshima and Nagasaki atomic bombings and of rare nuclear plant accidents (exacerbated by mass media), has led to very strict radiation protection regulation and standards that are potentially not scientifically justified. In the last three decades, numerous reports have documented both the lack of harmful and the presence of potentially beneficial biological effects induced by low dose radiation1-4. The major radiation health risk factor is the probability of cancer, estimated based on epidemiological studies of atomic bomb survivors receiving high or medium doses of radiation. Linear extrapolation of these data (so called linear-no-threshold or LNT model) is used to estimate risks of cancer at low doses. However, this approach has not received world-wide scientific acceptance and is heavily debated5.
It is evident that more studies are required to clarify this issue and possibly improve radiation protection standards. Such studies should involve chronic treatments (best approximation of environmental and occupational exposures), in vivo animal models (best for extrapolating effects to human) and such end-points as DNA damage rates, DNA repair and mutagenesis. It is known that DNA is the primary target for damaging radiation effects and incomplete or mis-repair may lead to mutagenesis and cancer development6.
DNA double-strand breaks (DSB) are one of the most deleterious types of DNA lesions and may lead to cell death and tumorigenesis7. It is, therefore, important to be able to reliably and accurately measure the level of DSB after exposure to low dose radiation and/or other stressors, such as chemical pollutants. One of the most sensitive and specific markers of DNA DSB is phosphorylated histone H2AX, called &#947;H2AX8, yet other markers and methods have been suggested9,10. It is estimated that thousands of H2AX molecules, in the vicinity of an induced DSB, are involved in the formation of &#947;H2AX enabling the detection of individual DSB by immunofluorescent labeling with an anti-&#947;H2AX antibody and fluorescence microscopy11. The response is very quick, reaching its maximum between 30 and 60 min. Evidence exists that &#947;H2AX facilitates repair of DNA DSB by attracting other repair factors to the sites of breaks and by modifying chromatin structure to anchor broken DNA ends and provide access for other repair proteins (reviewed in 12). Upon completion of repair of DNA DSB, &#947;H2AX gets de-phosphorylated and/or undergoes degradation, and newly synthesized H2AX molecules replace &#947;H2AX in the affected areas of chromatin10. Monitoring formation and loss of &#947;H2AX can, therefore, provide an accurate estimate of DNA DSB repair kinetics. This approach has been used to study repair of DSB in various human tumor cell lines irradiated with high doses of radiation and its rate and residual DSB levels have been shown to correlate with radiosensitivity13-15.
We modified this experimental approach and applied it to an in vivo mouse study to examine the effects of low doses of chronic &#947;- and &#946;-irradiation on DNA DSB levels and repair (Figure 1). Firstly, we demonstrate a method to perform a long-term chronic exposure of mice to &#946;-radiation either emitted by tritium (hydrogen-3) in the form of tritiated water (HTO) or as organically bound tritium (OBT) dissolved in drinking water. The two forms are expected to accumulate and/or distribute differently in the body and therefore, produce different biological effects. Both forms are potential hazards in nuclear industry. This treatment is paralleled by chronic exposure to &#947;-radiation at an equivalent dose rate to allow a correct comparison of the two radiation types, which is crucial for the evaluation of their relative biological effectiveness. Beta-radiation is composed of electrons, making it very different from the &#947;-radiation, high energy photons. Due to this difference, &#914;-radiation represents mostly internal health hazard and may produce different biological effects compared to &#947;-radiation. This complication resulted in significant controversies over the regulation of exposure to &#946;-radiation emitted by HTO. Thus, regulatory levels of HTO in drinking water for the public vary from 100 Bq/L in Europe to 75,000 Bq/L in Australia. It is, therefore, important to compare biological effects of HTO to equivalent doses of &#947;-radiation. Secondly, the rate of DNA DSB is measured in isolated splenocytes upon the completion of chronic exposures using immunofluorescently labeled &#947;H2AX detected by flow cytometry. This allows the evaluation of the extent of DNA damage inflicted by the in vivo exposures. However, it is sensible to expect that such low level exposures may not produce any detectable rates of DNA DSB; instead, some hidden changes/responses may be expected that would affect the cells&#8217; ability to repair DNA damage. These changes, if found, may be either stimulatory (producing a beneficial effect) or inhibitory (producing a deleterious effect). The proposed protocol allows revealing such changes by challenging the extracted splenocytes with a high dose of radiation that produces a significant amount of damage (e.g., 2 Gy producing approximately 50 DNA DSB per cell or a 3 - 5 fold increase in total &#947;H2AX level). Subsequently, the formation and loss of &#947;H2AX, reflecting the initiation and completion of DSB repair, is monitored by flow cytometry. In this way, not only basal and treatment-induced levels of DNA DSB can be measured, but also its potential effect on the cells&#8217; ability to respond and repair DNA damage induced by much higher stressor levels.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>HTO: tritiated water, [3H]</td>
			<td>locally obtained from a nuclear reactor&#160;</td>
			<td></td>
			<td>stock activity 3.7 GBq/mL;&#160; can be substituted with HTO from Perkin Elmer</td>
		</tr>
		<tr>
			<td>OBT: Alanine, L-[3-3H]: organically bound tritium (OBT)</td>
			<td>Perkin Elmer</td>
			<td>NET348005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>OBT: Glycine, [2-3H]: organically bound tritium</td>
			<td>Perkin Elmer</td>
			<td>NET004005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>OBT: Proline, L-[2,3-3H]: organically bound tritium (OBT)</td>
			<td>Perkin Elmer</td>
			<td>NET323005MC</td>
			<td>1mCi/mL (185 MBq)</td>
		</tr>
		<tr>
			<td>tritiated water, [3H] (HTO)</td>
			<td>Perkin Elmer</td>
			<td>NET001B005MC</td>
			<td>substitute for HTO of local origin&#160;&#160;</td>
		</tr>
		<tr>
			<td>GammaBeam 150 irradiator</td>
			<td>Atomic Energy of Canada Limited</td>
			<td>locally manufactured</td>
			<td>can be substituted with another g-radiation source of sufficiently low activity&#160;</td>
		</tr>
		<tr>
			<td>tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Fisher Scientific</td>
			<td>SH3025501</td>
			<td>Hyclone RPMI 1640 with L-Glutamine and HEPES 500mL</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Sigma Aldrich</td>
			<td>F1051-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-gH2AX antibody, clone JBW301</td>
			<td>Millipore</td>
			<td>05-636</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor-488 goat anti-mouse antibody</td>
			<td>Life Technologies (formerly Invitrogen)</td>
			<td>A21121</td>
			<td></td>
		</tr>
		<tr>
			<td>propidium iodine</td>
			<td>Sigma Aldrich</td>
			<td>P4864-10ML</td>
			<td>1mg/mL</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Commercial Alcohols</td>
			<td>P006-EAAN</td>
			<td>500ml bottles Absolute Ethanol</td>
		</tr>
		<tr>
			<td>1.5 mL tubes</td>
			<td>Fisher Scientific</td>
			<td>2682550</td>
			<td>microcentrifuge tubes</td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Fisher Scientific</td>
			<td>05-539-5</td>
			<td>sterile polypropylene centrifuge tubes</td>
		</tr>
		<tr>
			<td>liquid nitrogen</td>
			<td>Linde</td>
			<td>P110403</td>
			<td></td>
		</tr>
		<tr>
			<td>12 x 75 mm mL uncapped glass tubes</td>
			<td>Fisher Scientific</td>
			<td>K60B1496126</td>
			<td>disposable borosilicate glass tubes with plain end</td>
		</tr>
		<tr>
			<td>T25 flasks</td>
			<td>VWR</td>
			<td>CA15708-120</td>
			<td>nunc tisue culture 25ml flask (supplier no. 156340)</td>
		</tr>
		<tr>
			<td>scissors</td>
			<td>Fine Science Tools</td>
			<td>14068-12</td>
			<td>Wagner scissors 12cm sharp/sharp</td>
		</tr>
		<tr>
			<td>forceps, straight</td>
			<td>Fine Science Tools</td>
			<td>11008-13</td>
			<td>Semken forceps 13cm, straight</td>
		</tr>
		<tr>
			<td>forceps, curved</td>
			<td>Fine Science Tools</td>
			<td>11003-12</td>
			<td>Narrow Pattern forceps 12 cm, curved</td>
		</tr>
		<tr>
			<td>60 mm petri dishes</td>
			<td>VWR</td>
			<td>CA25382-100</td>
			<td>BD Falcon tissue culture dish 60x15mm</td>
		</tr>
		<tr>
			<td>cell strainers, 70 mm</td>
			<td>Fisher Scientific</td>
			<td>08-771-2</td>
			<td>Falcon cell strainers 50/case</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS recipe: 1 tablet dissolved in 200mL of deionized water, adjust pH to 7.4 if needed.</td>
			<td>Sigma Aldrich</td>
			<td>P4417-100TAB</td>
			<td>Phosphate Buffered Saline Tablets</td>
		</tr>
		<tr>
			<td>TBS recipe: to make 10X Stock,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30g TRIS HCl</td>
			<td>Sigma Aldrich</td>
			<td>T3253-1KG</td>
			<td>Trizma hydrochloride</td>
		</tr>
		<tr>
			<td>88g NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td>Sodium Chloride</td>
		</tr>
		<tr>
			<td>2g KCl</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td>Potassium Chloride</td>
		</tr>
		<tr>
			<td>Dissolve in 1L of deionized water, adjust pH to 7.4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring dna damage and repair in mouse splenocytes after chronic in vivo exposure to very low doses of beta- and gamma-radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Figure 2 shows examples of flow cytometry graphs expected for splenocytes prepared using the method described here. Cells are first gated based on the &#60;electronic volume-side scatter&#62; scatter plot (Figure 2A and 2D; electronic volume is equivalent to forward scatter). FL3/propidium iodine histograms (Figure 2B and 2E) confirm normal cell cycle distribution. Mean &#947;H2AX signal calculated using the FL1 channel confirms a &#62; 2-fold increase in DNA DSB in 2-Gy irradiated cells (Figure 2F) compared to the untreated control (Figure 2C). Next, we examined the sensitivity of the described method of measuring immunofluorescently labeled &#947;H2AX by flow cytometry in mouse splenocytes. Figure 3A shows &#947;H2AX levels, indicative of DNA DSB, in mouse splenocytes 1 hr after the ex vivo exposure to either low (0.1 Gy) or high (2 Gy) doses of &#947;-radiation relative to the untreated control. It can be seen that 0.1 Gy induced a slight increase in the DSB level and that increase was statistically significant. The strong 3-fold induction was expected and detected after the 2 Gy exposure. To validate the specificity of &#947;H2AX immunofluorescent labeling as described in the protocol, labeled cells were examined with a fluorescence microscope. The marked foci-like pattern observed indicates that the fluorescence signal originates from the individual DNA DSB within the chromatin (Figure 3B).
Representative results of the assessment of DNA DSB rate and their repair in splenocytes of mice exposed for one month to very low concentrations of tritiated water (10 kBq/L) are presented in Figure 4. It can be seen (Figure 4A) that although the treatment resulted in a 10% increase of the basal &#947;H2AX level, the change was not statistically significant (N = 5, one-sample Student&#8217;s t test). Furthermore, the measured formation and loss of &#947;H2AX after the challenging 2 Gy irradiation, representing the kinetics of DNA DSB repair, was not different between the cells from the control and HTO-treated mice (Figure 4B).
<img alt="Figure 1" src="/files/ftp_upload/52912/52912fig1.jpg" /> 
Figure 1.&#160;Experimental diagram to evaluate the effect of chronic in vivo irradiation on DNA damage and DNA repair. After in vivo treatment of mice with chronic irradiation for a desired period of time, mice are sacrificed and splenocyte cultures are established and exposed to a challenging irradiation. Splenocyte aliquots are collected and fixed at various times after the challenging dose. Levels of &#947;H2AX are then measured using immunofluorescent labeling and detection by flow cytometry. The plot on the bottom of the Figure is a schematic of expected data. Gamma-H2AX level drastically increases early after the challenging irradiation followed by a decrease back to the control value, if DNA repair is complete. DNA damage &#916; represents the damage produced due to the experimental treatment of mice and DNA repair &#916; represents the effect of the treatment of mice on DNA DSB repair capacity. 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52912/52912fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52912/52912fig2.jpg" /> 
Figure 2.&#160;Representative flow cytometry raw data. (A, D)&#160;Scatter plots and gating regions for splenocytes and cell debris and erythrocytes. (B, E)&#160;DNA histograms representing cell cycle distributions. (C, F)&#160;&#947;-H2AX fluorescence histogram and mean fluorescence values. Data in sets (A-C) were obtained from a representative untreated control spleen cells, whereas data in sets (D-F) were obtained from cells irradiated with 2 Gy &#947;-radiation and harvested 1 hr after the irradiation. 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52912/52912fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52912/52912fig3.jpg" /> 
Figure 3.&#160;Detection of &#947;H2AX by flow cytometry is sensitive and specific. (A)&#160;Splenocytes freshly isolated from male CBA mice were irradiated with either 0.1 or 2 Gy of &#947;-radiation, or sham-treated and allowed to develop &#947;H2AX response for 1 hr. Cells were then fixed, immunofluorescently labeled with anti-&#947;H2AX antibody and analyzed by flow cytometry. Normalized mean values &#177; SD are shown (N = 12). * and *** signify statistical difference with p&#160;&#60; 0.05 and p&#160;&#60; 0.001, respectfully (one-sample Student&#8217;s t-test). (B)&#160;Representative microphotographs of untreated control (UT) or 2 Gy irradiated splenocytes immunofluorescently labeled with anti-&#947;H2AX antibody. The foci-like pattern of green fluorescence confirms the specificity of the immunofluorescent labeling for &#947;H2AX. 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52912/52912fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52912/52912fig4.jpg" /> 
Figure 4.&#160;Representative results showing the lack of effect produced by 1-month exposure of mice to tritiated water (HTO) on DNA DSB level and repair. (A)&#160;Splenocytes were isolated from mice treated with 10 kBq/L of HTO in drinking water and control animals. The level of &#947;H2AX was measured using immunofluorescent labeling and flow cytometry as described in the protocol. Mean &#947;H2AX fluorescence levels relative to those of control &#177; SD are shown (N = 5). These data were generated from the t = 0 time-point that was used in Figure 3B for the evaluation of DNA DSB repair. (B)&#160;Isolated splenocytes were exposed to a challenging 2 Gy dose of &#947;-radiation and aliquots of cells were fixed at times t = 0, 1 and 24 hrs after the challenging irradiation. Mean fluorescence of &#947;H2AX relative to its own t = 0 control is shown &#177; SD (N = 5). 
<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52912/52912fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation at JoVE.com

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

measuring-dna-damage-repair-mouse-splenocytes-after-chronic-vivo

Nanyang Technological University

Research

JoVE Journal

Biology

11.0K Views.  Canadian Nuclear Laboratories. A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented. 

Video: Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the different aspects of visual, cross-modal and developmental plasticity along the mammalian visual system1-4.
Here, we demonstrate an elegant and straightforward technique for the removal of one or both eyes in the mouse, which is validated in mice of 20 days old up to adults. Briefly, a disinfected curved forceps is used to clamp the optic nerve behind the eye. Subsequently, circular movements are performed to constrict the optic nerve and remove the eyeball. The advantages of this technique are high reproducibility, minimal to no bleeding, rapid post-operative recovery and a very low learning threshold for the experimenter. Hence, a large amount of animals can be manipulated and processed with minimal amount of effort. The nature of the technique may induce slight damage to the retina during the procedure. This side effect makes this method less suitable as compared to Mahajan et al. (2011)5 if the goal is to collect and analyze retinal tissue. Also, our method is limited to post-eye opening ages (mouse: P10 - 13 onwards) since the eyeball needs to be displaced from the socket without removing the eyelids. The in vivo enucleation technique described in this manuscript has recently been successfully applied with minor modifications in rats and appears useful to study the afferent visual pathway of rodents in general.

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

The removal of eyes, also called enucleation, provides a useful strategy to study aspects of visual, cross-modal, and developmental plasticity along the mammalian visual system since it induces irreversible partial (monocular) or complete (binocular) vision loss. Here we describe a highly reproducible and straightforward approach to perform in vivo enucleation.

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

Measuring Glutathione-induced Feeding Response in Hydra

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy