Name: characterizing dna repair processes at transient and long-lasting double-strand dna breaks by immunofluorescence microscopy
Uploaded: 2018-06-08T13:30:00
Description: Watch this Scientific Journal Video about Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy at JoVE.com

We thank Joel Sanneman and Dr. Philine Wangemann of the Confocal Microscopy Core, funded by the Kansas State University College of Veterinary Medicine, for their support of efforts to develop this technique. pCBASceI was a gift from Maria Jasin (Addgene plasmid # 26477)30. U2OS DR-GFP cells were a kind gift from Maria Jasin18.

Please note, this protocol is written for U2OS cells containing an I-SceI recognition site18. The cells need not be U2OS but must contain the I-SceI site. The protocol may need to be adjusted (e.g., number of cells seeded and incubation times) depending on the type of cells used.
1. Defining the Kinetics of DSB Repair Complex Formation
<ol>
	<li>Grow U20S-DRGFP cells on a 10 cm tissue culture plate until 85&#8211;90% confluent.</li>
	<li>Remove the media and ...

<ol>
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The analysis of DNA damage repair in general and the repair of double-stranded DNA breaks specifically is an active area of research because its consequences span tumorigenesis to basic biology6,20. This manuscript details an approach that accurately dissects the contribution of RAD51 and &#947;-H2AX proteins to the resolution of DSBs through HR. Looking forward, this method can be used to elucidate additional functions of repair proteins at DSBs<sup class="xref"...

Each day, every cell in the human body is bombarded with an estimated 10,000 DNA lesions1. This existential threat puts us at risk for mutations, oncogenesis as well as cell death. To protect genome fidelity, mammalian cells have evolved to respond to DNA damage with a complex series of protein associations and modifications. This response is organized into multiple pathways, collectively known as the DNA damage response (DDR)2,3. The DDR consists of the accumulation of DNA repair proteins at DNA lesions, coordinated both temporally and spatially. DDR frequently induces cell cycle arrest to avoid the propagation or intensification of damage that can occur during the replication of damaged DNA2,4,5. In turn, it is also necessary for the cellular viability to turn off cell cycle arrest by disassociating repair complexes after repair has been completed.
Among the various types of DNA damage, DSBs are the most deleterious. Failure to repair DSBs can result in chromosome rearrangements or large-scale deletions such as the loss of entire chromosome arms. The repair of DSBs is divided into two pathways6,7,8. Homologous recombination (HR) requires a sister chromosome to use as a DNA template and thus is limited to late S and G2/M phases of the cell cycle9,10. Non-homologous end joining (NHEJ) does not have these restrictions but can cause small deletions when repairing DSBs11,12.
DSB repair specifically and the DDR in general are active areas of investigation. Despite being organized into conveniently separated pathways, there is a great deal of redundancy. Indeed, many proteins (BRCA1, BRCA2, and the RPA complex for example) are involved in multiple pathways13,14,15,16. The repair of a lesion by one pathway, can lead to a damage intermediate that must be repaired by another pathway14. The intertwining of these pathways, combined with their complex task of recruiting the right proteins to the correct place for the precise amount of time necessary, requires a multi-tiered regulatory process.
A recent report highlights the intricacies of DDR by demonstrating that repair complex formation, resolution, and localization can each separately be impaired17. The overall goal of the following protocol is to definitively dissect the ability of cells to repair DSB. Using immunofluorescence microscopy, accumulation of repair proteins at the sites of damage can be visualized at the representative time points following the induction of DSBs.
This technique has several advantages to commonly used approaches. Frequently, repair is investigated at single time points and incapable of representing the dynamic process of assembly and dissociation of repair complexes. Observing the full range of repair from the initial activation to the full resolution ensures that a delay in repair is not misidentified as complete inhibition. Conversely, it assures that the induction of a repair response that is unable to inactivate said response is not misidentified as the normal or the excessive activation.
Delayed protein complex formation and the mis-localization of repair proteins, however, may not be unambiguously distinguished with this approach. To determine if repair proteins are mis-localized versus delayed in their localization, a &#34;long-lasting&#34; DSB can be introduced through enzymatic cleavage of cellular DNA. The resulting lesion is recut each time it is repaired, resulting in a distinct large nuclear repair focus and removing the temporal restriction from recruitment. This can be achieved by modifying the existing approach with the use of a rare cutting endonuclease (e.g., I-SceI) to induce a long-lasting DSB. The longevity of DSBs enables the visualization of elusive repair proteins by immunofluorescence microscopy. The enhanced abundance could also improve the visualization when detection is hindered by limitation in the antibody quality, a feature that could be useful when lesser studied proteins are identified as having an impact on DNA repair.
Notably, we provide explicit instructions for a free image processing and analysis software (e.g., ImageJ). This removes a major financial barrier in image analysis, opening the interrogation of DNA damage repair to a wider audience.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>12mm Coverslips</td>
			<td>VWR</td>
			<td>89015-725</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (PFA)</td>
			<td>ThermoFisher Scientific</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>VWR</td>
			<td>82050-892</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well glass bottom plate</td>
			<td>Cellvis</td>
			<td>P96-1.5H-N</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-H2AX Alexa488</td>
			<td>EMD Millipore</td>
			<td>05-636-AF488</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Rad51 (D4B10)&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>8875S</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-formats plugin for ImageJ</td>
			<td>National Institute of Health (NIH)&#160;</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/plugins/index.html</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>VWR</td>
			<td>97061-416</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, High Glucose</td>
			<td>ThermoFisher Scientific</td>
			<td>12100046</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15576-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>VWR</td>
			<td>89510-194</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG Alexa594</td>
			<td>ThermoFisher Scientific</td>
			<td>&#160;A-11012&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>sigma-Aldrich</td>
			<td>216763-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>National Institute of Health (NIH)&#160;</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>I-SceI Expression Vector</td>
			<td>Addgene</td>
			<td>26477</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail Polish- Insta Dri</td>
			<td>Sally and Hansen</td>
			<td>Clearly Quick (103)</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Bio Basic</td>
			<td>PD8117</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Reagent</td>
			<td>Life Technologies</td>
			<td>P36930</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TurboFect Transfection Reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>R0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing dna repair processes at transient and long-lasting double-strand dna breaks by immunofluorescence microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Figure 1 depicts the selection of the correct noise discrimination for maxima/foci quantification using ImageJ. The merged images of DAPI and the repair protein of interest are on the left panel. Figure 1A shows a noise discrimination of 90 and marks the correct number of foci. Nuclei on the edge (depicted with a pink arrow) and foci outside the nuclei (depicted with a yellow arrow) are not counted during the quantification. <str...

Watch this Scientific Journal Video about Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy at JoVE.com

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

This method can help answer key questions in the DNA repair field such as how DNA repair complex formation, localization, and resolution are regulated. The main advantage of this technique is that it can unambiguously detect changes and repair complex localization and kinetics. In addition to these undergraduate researchers in my lab, three graduate students will perform the experiments.That will be Changkun Hu, Dalton Dacus, and Stephen Walterhouse. To begin, grow U2OS/DR-GFP cells on a 10 centimeter tissue culture plate until they are 85 to 90%confluent. Then remove the medium, add three milliliters of EDTA, and incubate the cells at room temperature for three minutes.Replace the EDTA with one milliliter of trypsin and incubate the cells at 37 degrees Celsius for five minutes. Then add five milliliters of DMEM to neutralize the trypsin. After counting the cells, seed six times 10 to the third cells per well in 200 microliters of medium in a glass bottom 96-well plate.Alternatively, seed four times 10 to the six wells in eight milliliters of medium on 12 millimeter cover slips placed in a 10 centimeter plate, then grow the cells over night at 37 degrees Celsius and 5%carbon dioxide. To induce double strand breaks, or DSBs, replace the medium with hydrogen peroxide diluted to a concentration of 25 micromolar with DMEM plus 10%FBS and incubate the plate at 37 degrees Celsius for one hour. Wash the cells three times with 1x PBS, then add fresh DMEM.To fix the cells after incubating them, use PBS to wash the plate three times. Then use a needle and forceps to carefully remove a cover slip from the 10 centimeter plate for each time point and place it in a single well of a 24-well plate. Add 4%PFA and incubate the cells for 15 minutes.Then us PBS to wash the cover slips three times. To permeabilize the cells, add 0.5%non-ionic detergent and PBS. Incubate the cells at room temperature for 15 minutes.Then wash the cells again with PBS three times. Next add 3%BSA and 0.1%non-ionic detergent diluted in PBS to the cover slips in a 24-well plate and incubate the plate at room temperature for one hour. Then add primary antibodies that target the repair protein of interest and incubate the samples at room temperature for one hour.After washing the cells with PBS three times, add DAPI and incubate the plates at room temperature for five minutes. Then incubate the samples with secondary antibodies. After washing the cells as before, use mounting reagent to mount the cover slips cell side down onto slides.Carefully press on the cover slips and use a wipe to soak up any excess mounting reagent. Seal the cover slips with fast drying transparent nail polish and ensure a complete seal of the cover slip with the slide. Take confocal microscope images by focusing on the DAPI channel.To define the nuclei in immunofluorescence images, open the confocal images by dragging the image file into the ImageJ window or by selecting bio-formats importer from the plugin toolbar in ImageJ. Then select split channels. Under color options select colorized from the dropdown menu.Select OK to open the DAPI and histone H2AX images as separate windows and choose the DAPI image. Then select the image toolbar in the adjust threshold option to select the nuclei. Next, adjust the image threshold by sliding the bottom bar of the threshold window completely to the right.Adjust the top bar until the nuclei appear completely red with distinct outlines and the background is black. Then select the analyze toolbar and the analyze particles option. Input the minimum size of the nucleus.From the show dropdown menu select outlines then select the add to manager option. Click OK to open an ROI manager window. From the ROI manager window assign numbers to the nuclei by selection.To use ImageJ to quantify H2AX foci and immunofluorescence microscopy images select the H2AX foci window. Then select process toolbar and choose find maxima to find foci within the nuclei. From the output type dropdown menu choose the single points option and select the preview point selection to visualize the maxima foci.Then input a noise tolerance value depending on the florescence intensity of the image. From the ROI manager window select the nuclei for which H2AX foci must be quantified. Select measure to measure the number of maxima foci within the selected nuclei.Copy the raw intensity values and paste them into software for data analysis. After seating cells on 96-well plates with cover slips as demonstrated earlier in this video, induce double strand breaks by using a lipid-based transfectionary agent to transfect cells with the I-SceI expression vector. To acquire imagine after washing vix permeabilized and blocked cells as shown earlier, add antibodies against gamma H2AX and repair protein of interest.With 1x PBS wash the 96-well plate cover slips three times. Then incubate the 96-well plate cover slips with secondary antibodies. Finally, after identifying the cells that have a large nuclear gamma H2AX focus, manually count those that also show colocalization with the repair protein of interest.This figure shows a noise discrimination of 90 and marks the correct number of foci. Nuclei on the edge and foci outside the nuclei are not counted during the quantification. The panel shown here depicts a low noise tolerance of 50.Numerous maxima are identified outside the nucleus and within the nucleus. A high noise tolerance of 220 is found in this cell. Only a single foci was detected.Colocalization of a gamma H2AX focus and a repair protein of interest is depicted in this figure. An untransfected cell is shown here, and a cell with non-nuclear gamma H2AX focus is shown in the upper right. At higher magnification a clearer picture of a true interior focus and a non-nuclear or exterior focus can be distinguished.Finally, an example of a nuclear gamma H2AX focus without colocalization of a repair protein is shown here. Once mastered, this technique can be done in a few days if it is performed properly. Following this procedure other methods like co-mutor precipitation can be performed in order to identify interactions among repair proteins of interest.After watching this video, you should have a good understanding of how to observer the formation and resolution of DNA repair complexes by inducing transient double strand breaks in DNA. You will also learn how to unambiguously distinguish mislocalization of repair factors from delayed recruitment by inducing long-last lesions.

characterizing-dna-repair-processes-at-transient-long-lasting-double

Research

JoVE Journal

Cancer Research

Cell-Free DNA Integrity Analysis in Urine Samples

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.
A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of &#947;H2AX and 53BP1, the combined analysis of &#947;H2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on &#947;H2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the &#947;H2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of &#947;H2AX and 53BP1.

Immunofluorescence Microscopy of &amp;#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

This manuscript provides a protocol for the analysis of DNA double-strand breaks by immunofluorescence microscopy of &#947;H2AX and 53BP1.

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Quantifying Replication Stress in Ovarian Cancer Cells Using Single-Stranded DNA Immunofluorescence

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, transcription-replication conflicts, or amplified oncogenes, inevitably resulting in the generation of single-stranded DNA (ssDNA). Quantifying ssDNA, therefore, presents an opportunity to assess the level of replication stress in different cell types and under various DNA-damaging conditions or treatments. Emerging evidence also suggests that ssDNA can be a predictor of responses to chemotherapeutic drugs that target DNA repair. Here, we describe a detailed immunofluorescence-based methodology to quantify ssDNA. This methodology involves labeling the genome with a thymidine analog, followed by the antibody-based detection of the analog at the chromatin under non-denaturing conditions. Stretches of ssDNA can be visualized as foci under a fluorescence microscope. The number and intensity of the foci directly co-relate with the level of ssDNA present in the nucleus. We also describe an automated pipeline to quantify the ssDNA signal. The method is rapid and reproducible. Furthermore, the simplicity of this methodology makes it amenable to high-throughput applications such as drug and genetic screens.

Here, we describe an immunofluorescence-based method to quantify the levels of single-stranded DNA in cells. This efficient and reproducible method can be utilized to examine replication stress, a common feature in several ovarian cancers. Additionally, this assay is compatible with an automated analysis pipeline, which further increases its efficiency.

Replication stress is a hallmark of several ovarian cancers. Replication stress can emerge from multiple sources, including double-strand breaks, ...

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Diagnosis of Vitreoretinal Lymphoma Using Cell-Free DNA

Cell-Free DNA Extraction of Vitreous and Aqueous Humor Specimens for Diagnosis and Monitoring of Vitreoretinal Lymphoma

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To diagnose VRL, specimens such as vitreous humor and, more recently, aqueous humor are collected. Diagnostic testing for VRL on these specimens includes cytology, flow cytometry, and molecular testing. However, both cytopathology and flow cytometry, along with molecular testing using cellular DNA, necessitate intact whole cells. The challenge lies in the fact that vitreous and aqueous humor typically have low cellularity, and many cells get destroyed during collection, storage, and processing. Moreover, these specimens pose additional difficulties for molecular testing due to the high viscosity of vitreous humor and the low volume of both vitreous and aqueous humor. This study proposes a method for extracting cell-free DNA from vitreous and aqueous specimens. This approach complements the extraction of cellular DNA or allows the cellular component of these specimens to be utilized for other diagnostic methods, including cytology and flow cytometry.

Author Spotlight: Advancing VRL Diagnosis Using Cell-Free DNA Extraction from Vitreous Humor 

A procedure to extract cell-free DNA from vitreous and aqueous humor to perform molecular studies for diagnosing vitreoretinal lymphoma is established here. The method offers the ability to concurrently extract DNA from the cellular component of the sample or to reserve it for ancillary testing.

Vitreoretinal lymphoma (VRL) represents an aggressive lymphoma, often categorized as primary central nervous system diffuse large B-cell lymphoma. To ...