The authors wish to thank Drs. Philina S. Lee and Alan D. Grossman for initially training L.A.S. in fluorescence microscopy. The authors also thank Drs. Melanie Berkmen and Hajime Kobayashi for help and tips for imaging. This work was supported by start-up funds from the College of Literature, Science &amp; Arts and from the Department of Molecular, Cellular, and Developmental Biology at the University of Michigan.

Growing cultures of cells for microscopy
1. One or two days prior to imaging, prepare the B. subtilis strain containing the translational fusion protein you wish to visualize. Trial rounds of steps 1A and 1B will be necessary to determine which growth condition provides the best images of your strain. In most cases, cells should be imaged during exponential growth phase.
<ol class="ualpha">
<li>For some B. subtilis strains (in particular, strains that grow poorly due to the integration of the translational fusion between the gene of interest and GFP at its endogenous locus), initial growth for two days prior to imaging is necessary for high quality images. On the first day, streak the B. subtilis strain to be imaged on LB agar with selective antibiotic(s) and incubate overnight at 30&deg;C. On the second day, inoculate 100 &mu;l of 0.85% saline with a single colony and perform three 10-fold serial dilutions on LB agar plates (with selective antibiotics), plating 100 &mu;L of each dilution, followed by incubation overnight at 30&deg;C. On the third day, select the dilution plate with light confluent growth of cells. Light confluent growth on a plate will provide the growth medium with a healthy, exponentially growing starter inoculum to potentially yield excellent imaging results (see step 4). This step is particularly useful for the highest quality membrane imaging with the stain FM4-64. </li>
<li>Other B. subtilis strains may simply be applied to selective LB agar medium for single colonies with a sterile stick the day prior to imaging and incubate overnight at 30&deg;C. The following day, cells on this plate will be sufficient to use as an inoculum for imaging (see step 4). NOTE: For B. subtilis, overnight cultures in liquid medium are not appropriate due to a combination of spore formation and quorum responses, which will cause an extended period of lag phase growth 1.</li>
</ol>
2. On the morning of imaging, place 10 mL of S750 1-3 medium into a 125 mL sterile Erlenmeyer flask. 
3. Resuspend the cells in up to 2 mL of S750 medium from the LB agar plate with single colonies or the light confluent cells to obtain an inoculum. 
4. Add enough S750 medium-cell inoculum to the Erlenmeyer flask containing 10 mL S750 medium for a culture with an initial optical density measured at 600 nm (OD600) of ~0.08 to 0.1. At this step it is important to have at least a 10-fold air to medium ratio. Thus, we use 10 to 12.5 mL of inoculated medium in a 125 mL Erlenmeyer flask. 
5. Grow each culture at 30&deg;C until the culture reaches an OD600 ~0.5-0.8. Shaking water baths are preferred with revolutions per minute from 150-200. Cultures challenged with DNA damaging or mismatch inducing agents should be at an early exponential growth phase OD600 such that the cells will reach the target OD600 with the additional period of growth that is required for treatment (see Table 1) 
 For example, 2-aminopurine requires one hour of treatment, and therefore a culture treated with 2-aminopurine should be at OD600~0.3-0.4, such that the one hour of treatment/growth results in an OD600~0.5-0.8, whereby cells are ready for imaging. 
Sample Preparation
1. Pipette 300 &mu;l of cultured cells into a 1.5 mL microcentrifuge tube.
2. If imaging of cellular membranes is desired, add 1:1000 dilution of FM4-64 membrane stain (Invitrogen) to each sample from a 1 mg/mL stock solution. A titration of FM4-64 may be required to achieve the desired fluorescence signal. A typical titration range is 1:100, 1:1000, and 1:10,000.
3. Allow the samples to sit at room temperature for up to ten minutes or while the slide is being prepared with 1% agarose in 1X Spizizens medium as described below (see step 1 of "slide preparation").
4. If concentration of cells is required, we prefer filtering cells using a 0.2 &mu;m filter with a vacuum apparatus. Cells can then be gently washed from the surface of the filter with PBS, another appropriate buffer or medium prior to visualization.
Slide Preparation
1. Prepare 1% agarose with 1X Spizizens. Add 5 mL of 10X Spizizens stock to 45 ml ddH2O along with 0.5 g agarose, and microwave to melt. Hot molten agarose is difficult to pipette, and solidified agarose will not enter the pipette tip. The agarose should be equilibrated in a water bath to a temperature of ~55-65&deg;C for it to enter the pipette tip without solidifying within the tip.
2. Draw 20 &mu;l of Spizizens with 1% melted agarose into the pipette tip. Pipette a single drop (approximately 1-2 &mu;L) of the agarose solution into each slide well (we use 15-well slides, see Table 2) to form shallow agarose pads. The agarose will solidify immediately. Slides must also be made immediately prior to application of the cells. If the agarose pads are left at room temperature alone, they can dehydrate within minutes and become unusable. 
Agarose pads should be centered and fill each well but have minimum height to avoid disrupting the coverslip; if droplets are too high (bubble-like) or if the sample spreads beyond the well boundary, simply swipe the well clean with a Kimwipe and apply a new agarose pad. 
3. Apply the entire 300 &mu;l sample (see step 6) to the slide, distributed on the top of each agarose pad. This amount of cells should be sufficient to cover each well on the slide.
4. Allow the slide loaded with cells to sit at room temperature (23&deg;C) for approximately ten minutes. This step will allow for the cells to settle on the agarose pad becoming immobile and ready for imaging. For specialized needs this temperature may need to be changed, for example, when performing experiments that require a temperature shift4.
5. Aspirate away the excess medium from each well without removing the pads themselves.
6. Apply the coverslip to the slide, pressing firmly and evenly, but gently, across the area of the slide. Check to make sure that the cover slip is against the slide and that the cover slip is not raised above the slide.
Visualization of cells
1. Many different fluorescence microscopes will work well. It is critical to have a 100X oil immersion lens. The Simmons Lab uses:
<ul>
 <li>Olympus BX61 microscope equipped with 1,45 NA TIRFM 100X oil immersion objective lens</li>
 <li>Hamamatsu ORCAR2 CCD cooled camera</li>
 <li>Lumen 200 arc metal (Prior) light source. </li>
 <li>For detection of GFP (FITC), filter excitation 460-500 and emission 510-560</li>
 <li>For detection of FM4-64 (TRITC), filter excitation 510-560 and emission 572-648. </li>
 <li>Images were captured using SlideBook 4.2 (Figure 1) </li>
</ul>
2. Bring the cells into focus using white light.
3. Capture the image using appropriate filters for each fluorophore. 
Representative Results
Representative images are shown (Figure 1). GFP foci should be well defined, while FM4-64 staining should be bright and clear 4-7. GFP or membrane images can be pseudo-colored with any color, to allow for the highest quality image to be presented without losing any data.
<img src="/files/ftp_upload/1736/1736fig1.jpg" alt="Figure 1" /> Figure 1: Representative GFP images of B. subtilis translational fusion proteins. GFP proteins are shown in green, while FM4-64 membrane staining is shown in red. The white scale bar indicates 3 &mu;m. (A) MutS-GFP [relevant genotype: mutS-gfp (spc), amyE::PspacmutL (cat)] in the presence of 600 &mu;g/mL 2-aminopurine and 1 mM IPTG. FM4-64 signal is not presented in order to more clearly show the MutS-GFP foci. (B) MutL-GFP [relevant genotype: mutL::mutL-GFP (spc)] in the presence of 2-aminopurine. (C) DnaX-GFP [relevant genotype: dnaX::dnaX-GFP (spc)]. (D) RecA-GFP [relevant genotype: recA::recA-GFP (spc)] in the presence of 20 ng/mL mitomycin C. (E) TagC-GFP [relevant genotype: tagC::tagC-GFP (spc)] in the presence of 1 &mu;g/mL mitomycin C. 

<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Treatment /concentration or amount</td>
 <td>Function</td>
 <td>Time of incubation</td>
 </tr>
 <tr>
 <td>Mitomycin C (MMC) [20 to 150 ng/mL]</td>
 <td>DNA alkylating agent</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>2-aminopurine (2-AP) [600 &mu;g/mL]</td>
 <td>mismatch inducing</td>
 <td>1 hour</td>
 </tr>
 <tr>
 <td>Hydroxyurea (HU)</td>
 <td>depletes dNTPs</td>
 <td>3 hours</td>
 </tr>
 <tr>
 <td>ultraviolet light (UV) 20 J/m2</td>
 <td>Thymine-thymine dimers and 6-4 photoproducts</td>
 <td>varies depending on source, usually 20 seconds</td>
 </tr>
 <tr>
 <td>grays (ionizing radiation) 5 to 100 Gy</td>
 <td>Double strand breaks, single strand breaks and base damage sites</td>
 <td>varies depending on source </td>
 </tr>
</tbody>
</table>
Table 1. Summary of DNA damaging and mismatch inducing treatments

<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Multitest slide, 15-well</td>
 <td>MP Biomedicals</td>
 <td>6041505E</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscope Cover Glass</td>
 <td>Fisher</td>
 <td>12-544-B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Fisher</td>
 <td>BP160-500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Mitomycin C</td>
 <td>Sigma</td>
 <td>M0503-2MG</td>
 <td>mutagen, wear gloves</td>
 </tr>
 <tr>
 <td>2-Aminopurine</td>
 <td>Sigma</td>
 <td>A3509-250MG</td>
 <td>gloves</td>
 </tr>
 <tr>
 <td>Hydroxyurea</td>
 <td>Sigma</td>
 <td>H8627-25G</td>
 <td>gloves</td>
 </tr>
 <tr>
 <td>FM4-64</td>
 <td>Invitrogen</td>
 <td>T13320</td>
 <td>light sensitive</td>
 </tr>
</tbody>
</table>
Table 2. List of specific reagents and equipment:

<ol>
	<li>Hardwood, C. R., Cutting, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biological Methods for Bacillus. , (1990).</li><li>Berkmen, M. B., Grossman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+organization+of+the+Bacillus+subtilis+replication+cycle.">Spatial and temporal organization of the Bacillus subtilis replication cycle.</a> Mol. Microbiol. 62, 57-71 (2006).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clp+and+Lon+proteases+occupy+distinct+subcellular+positions+in+Bacillus+subtilis.">Clp and Lon proteases occupy distinct subcellular positions in Bacillus subtilis.</a> J. Bacteriol. 190, 6758-6768 (2008).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+is+required+for+the+RecA+localization+response+to+DNA+damage+in+Bacillus+subtilis.">Replication is required for the RecA localization response to DNA damage in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U S A. 104, 1360-1365 (2007).</li><li>Simmons, L. A., Davies, B. W., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=b+Clamp+Directs+Localization+of+Mismatch+Repair+in+Bacillus+subtilis.">b Clamp Directs Localization of Mismatch Repair in Bacillus subtilis.</a> Mol. Cell. 29, 291-301 (2008).</li><li>Simmons, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+responses+to+double-strand+breaks+between+Escherichia+coli+and+Bacillus+subtilis+reveals+different+requirements+for+SOS+induction.">Comparison of responses to double-strand breaks between Escherichia coli and Bacillus subtilis reveals different requirements for SOS induction.</a> J. Bacteriol. 191, 1152-1161 (2009).</li><li>Smith, B. T., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+mismatch+repair+in+bacterial+cells.">Visualization of mismatch repair in bacterial cells.</a> Mol. Cell. 8, 1197-1206 (2001).</li></ol>

Trial and error are required to find exposure conditions for the highest quality images for each strain; we find that 1 millisecond is appropriate for white light images, while exposures of 100 to 2000 ms are appropriate for GFP (FITC) and FM4-64 (TRITC) images. Exposure time will vary depending on the imaging equipment used. We recommend the use of one strain per 15-well microscope slide for the simplest imaging, as pad quality and diffusion of cells from the pad border could complicate strain differentiation if multipl...

Specific solution recipes1:
10x S750 salts 
 0.5 M MOPS 
 100 mM Ammonium Sulfate 
 50 mM Potassium Phosphate Monobasic 
 Filter sterilize, and wrap S750 media in foil prior to storage
100x Metals 
 0.2 M MgCl2 
 70 mM CaCl2 
 5 mM MnCl2 
 0.1 mM ZnCl2 
 100 &mu;g/mL Thiamine HCl 
 2 mM HCl 
 0.5 mM FeCl3* 
 dH2O to final volume 
 *FeCl3 should be added last, to prevent precipitation. 
 After filter sterilization, wrap 100x Metal solution in foil.
S750 media 
 1x S750 salts 
 1x Metal 
 1% Glucose 
 0.1% Glutamate 
 40 &mu;g/mL Tryptophan 
 40 &mu;g/mL Phenylalanine 
 distilled H2O to final volume
10x Spizizens (grams/L) 
 151.4 mM Ammonium Sulfate (20g/L) 
 803.8 mM Potassium Phosphate Monobasic (140g/L) 
 440.9 mM Potassium Phosphate Dibasic (60g/L) 
 34.0 mM Sodium Citrate (10g/L) 
 16.6 mM MgSO4 (2g/L) 
 dH2O to final volume 
 Filter sterilize

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.

Watch this Scientific Journal Video about Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis at JoVE.com

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.

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 ...

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10.9K Views.  University of Michigan-Ann Arbor. A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

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

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

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 ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

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.

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.

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

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 ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...