Name: assessment of global dna double-strand end resection using brdu-dna labeling coupled with cell cycle discrimination imaging
Uploaded: 2021-04-28T15:00:00
Description: Watch this Scientific Journal Video about Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging at JoVE.com

We thank Marie-Christine Caron for outstanding technical advice. This work is supported by funding from Canadian Institutes of Health Research J.Y.M (CIHR FDN-388879). J.-Y.M. holds a Tier 1 Canada Research Chair in DNA Repair and Cancer Therapeutics. J.O&#39;S is an FRQS PhD student fellow, and S.Y.M is a FRQS postdoctoral fellow.

1. Cell culture, treatments, and coverslip preparation
NOTE: All cell plating, transfections, and treatments, aside from irradiation, should take place under a sterile cell-culture hood.
<ol>
	<li>Day 1
	<ol>
		<li>In a 6-well plate, place a single coverslip in each well for as many conditions as needed. Plate ~150,000 HeLa cells for transfection or drug treatment, as desired. 
		NOTE: If transfecting, it is recommended to do a reverse transfection at the time of plating, or it is possi...

<ol>
	<li>Mouw, K. W., Goldberg, M. S., Konstantinopoulos, P. A., D'Andrea, 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=DNA+damage+and+repair+biomarkers+of+immunotherapy+response.">DNA damage and repair biomarkers of immunotherapy response.</a> Cancer Discovery. 7 (7), 675-693 (2017).</li><li>Scully, R., Panday, A., Elango, R., Willis, N. A. <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-strand+break+repair-pathway+choice+in+somatic+mammalian+cells.">DNA double-strand break repair-pathway choice in somatic mammalian cells.</a> Nature Reviews Molecular Cell Biology. 20 (11), 698-714 (2019).</li><li>Chang, H. H. Y., Pannunzio, N. R., Adachi, N., Lieber, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-homologous+DNA+end+joining+and+alternative+pathways+to+double-strand+break+repair.">Non-homologous DNA end joining and alternative pathways to double-strand break repair.</a> Nature Reviews Molecular Cell Biology. 18 (8), 495-506 (2017).</li><li>Heyer, W. D., Ehmsen, K. T., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+homologous+recombination+in+eukaryotes.">Regulation of homologous recombination in eukaryotes.</a> Annual Review of Genetics. 44, 113-139 (2010).</li><li>Ronato, D. 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=Limiting+the+DNA+double-strand+break+resectosome+for+genome+protection.">Limiting the DNA double-strand break resectosome for genome protection.</a> Trends in Biochemical Science. 45 (9), 779-793 (2020).</li><li>Hu, Y., 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=PARP1-driven+poly-ADP-ribosylation+regulates+BRCA1+function+in+homologous+recombination-mediated+DNA+repair.">PARP1-driven poly-ADP-ribosylation regulates BRCA1 function in homologous recombination-mediated DNA repair.</a> Cancer Discovery. 4 (12), 1430-1447 (2014).</li><li>Satoh, M. S., Lindahl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+poly(ADP-ribose)+formation+in+DNA+repair.">Role of poly(ADP-ribose) formation in DNA repair.</a> Nature. 356 (6367), 356-358 (1992).</li><li>Sugimura, K., Takebayashi, S., Taguchi, H., Takeda, S., Okumura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP-1+ensures+regulation+of+replication+fork+progression+by+homologous+recombination+on+damaged+DNA.">PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA.</a> Journal of Cell Biology. 183 (7), 1203-1212 (2008).</li><li>Bryant, H. 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=Specific+killing+of+BRCA2-deficient+tumours+with+inhibitors+of+poly(ADP-ribose)+polymerase.">Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase.</a> Nature. 434 (7035), 913-917 (2005).</li><li>Farmer, H., 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=Targeting+the+DNA+repair+defect+in+BRCA+mutant+cells+as+a+therapeutic+strategy.">Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy.</a> Nature. 434 (7035), 917-921 (2005).</li><li>Zhou, P., Wang, J., Mishail, D., Wang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advancements+in+PARP+inhibitors-based+targeted+cancer+therapy.">Recent advancements in PARP inhibitors-based targeted cancer therapy.</a> Precision Clinical Medicine. 3 (3), 187-201 (2020).</li><li>Noordermeer, S. M., van Attikum, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP+inhibitor+resistance:+a+tug-of-war+in+BRCA-mutated+cells.">PARP inhibitor resistance: a tug-of-war in BRCA-mutated cells.</a> Trends in Cell Biology. 29 (10), 820-834 (2019).</li><li>Caron, M. C., 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=Poly(ADP-ribose)+polymerase-1+antagonizes+DNA+resection+at+double-strand+breaks.">Poly(ADP-ribose) polymerase-1 antagonizes DNA resection at double-strand breaks.</a> Nature Communications. 10 (1), 2954 (2019).</li><li>Zhou, Y., Caron, P., Legube, G., Paull, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+DNA+double-strand+break+resection+intermediates+in+human+cells.">Quantitation of DNA double-strand break resection intermediates in human cells.</a> Nucleic Acids Research. 42 (3), 19 (2014).</li><li>Raderschall, E., Golub, E. I., Haaf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+foci+of+mammalian+recombination+proteins+are+located+at+single-stranded+DNA+regions+formed+after+DNA+damage.">Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage.</a> Proceedings of the National Academy of Sciences of the United States of America. 96 (5), 1921-1926 (1999).</li><li>Sartori, A. 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=Human+CtIP+promotes+DNA+end+resection.">Human CtIP promotes DNA end resection.</a> Nature. 450 (7169), 509-514 (2007).</li><li>Huertas, P., Cruz-Garc&#237;a, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+molecule+analysis+of+resection+tracks.">Single molecule analysis of resection tracks.</a> Methods in Molecular Biology. 1672, 147-154 (2018).</li><li>Soniat, M. M., Myler, L. R., Kuo, H. -. C., Paull, T. T., Finkelstein, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RPA+phosphorylation+inhibits+DNA+resection.">RPA phosphorylation inhibits DNA resection.</a> Molecular Cell. 75 (1), 145-153 (2019).</li></ol>

This paper describes a method that makes use of IF staining to measure variations in DNA resection in cellulo. The current standard for observing an effect on DNA resection is through RPA staining; however, this is an indirect method that may be influenced by DNA replication. Previously, another BrdU incorporation-based DNA resection IF technique has been described for classifying the resulting intensities in BrdU-positive and BrdU-negative cells. This method allowed for cells that are not undergoing HR to be co...

Modulation of DNA repair factors is an ever-evolving method for cancer therapy, particularly in DNA DSB repair-deficient tumor environments. The inhibition of specific repair factors is one of the ingenious strategies used to sensitize cancer cells to DNA-damaging agents. Decades of research led to the identification of various mutations of DNA repair genes as biomarkers for therapeutic strategy choices1. Consequently, the DNA repair field has become a hub for drug development to ensure a wide range of treatments, empowering the personalized medicine concept. 
DSBs are repaired by two main pathways: NHEJ and HR2. The NHEJ pathway is error-prone, rapidly ligating the two DNA ends with little to no DNA end-processing and involving the protein kinase (DNA-PKcs), the Ku70/80 complex, 53BP1, and RIF1 proteins3. In contrast, HR is a faithful mechanism initiated by BRCA14. An essential step in HR repair is the DNA-end resection process, which is the degradation of the broken ends leading to single-stranded (ss) DNA with 3'-OH ends. BRCA1 facilitates the recruitment of the downstream proteins that form the resectosome MRN/RPA/BLM/DNA2/EXO1, which are involved in the 5' to 3' DNA resection5. 
The initial end-resection is accomplished through the endonuclease activity of MRE11, allowing for further processing by the DNA2 and EXO1 nucleases. The generated ssDNA overhangs are quickly coated by Replication Protein A (RPA) to protect them from further processing. Subsequently, BRCA2, PALB2, and BRCA1 engage to mediate the displacement of RPA and the assembly of the RAD51 nucleofilament required for homology-directed repair mechanism. A fine balance between the usage of NHEJ and HR is necessary for the optimal maintenance of genomic integrity. The pathway choice depends on the cell cycle phase. HR is preferentially used during the S to G2 phases wherein DNA resection is at the highest level, and the sister chromatids are available to ensure proper repair.
Poly (ADP-ribose) polymerase 1 (PARP-1) is one of the earliest proteins recruited to the DSB. It regulates both resection activity and the assembly of downstream effectors involved in the NHEJ5,6. PARP-1 is also required for DNA single-stranded break repair during replication7,8. Due to its important role in DNA repair, PARP inhibitors (PARPi) are used as cancer therapies. In several HR-deficient cancers, PARPi treatment leads to a synthetic lethal response due to the incapacity of HR-deficient cells to repair the accumulated damage via an alternative pathway9,10. There are currently four FDA approved PARPi: Olaparib, Rucaparib, Niriparib, and Talazoparib (also called BMN 673), which are used for various breast and ovarian cancer treatments11. However, PARPi resistance is common, and one potential cause arises through the reacquisition of HR proficiency12. Loss or inhibition of PARP-1 in the presence of irradiation dysregulates the resectosome machinery, leading to the accumulation of longer ssDNA tracts13. Therefore, an in-depth study of DNA resection in vivo is critical for a clearer understanding of the DNA repair pathways and the subsequent development of new strategies to treat cancer and to overcome PARPi resistance. 
There have been several methods employed to detect DNA resection events5. One such method is the classical IF-based technique allowing for indirect staining and visualization of the resected DNA after stress-induced DSB by using an anti-RPA antibody. Labelling genomic DNA with 5-bromo-2&#8242;-deoxyuridine (BrdU) and detecting only ssDNA is a direct measurement of DNA resection events. It circumvents the monitoring of RPA, which is involved in multiple cellular processes such as DNA replication. In the method described here, cells incubated with BrdU for a single cell cycle allow BrdU to be incorporated into one strand of the replicating cellular DNA. Following resection, IF staining is performed under conditions allowing wherein BrdU detection only in the ssDNA form, with the use of an anti-BrdU antibody. This antibody can only access exposed BrdU nucleotides and will not detect those integrated into double-stranded DNA. Using fluorescence microscopy, the resected DNA can be visualized in the form of punctate BrdU/ssDNA foci. The nuclear intensity of these foci can be used as a readout to quantify resection following DNA damage. This paper describes step-by-step the processes of this method, which can be applied to most mammalian cell lines. This method should be of broad utility as a simple way of monitoring DNA end resection in cellulo, as a proof of concept.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa 568 goat anti-rabbit</td>
			<td>Molecular probes</td>
			<td>A11011</td>
			<td>1:800</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse</td>
			<td>Molecular probes</td>
			<td>A11001</td>
			<td>1:800</td>
		</tr>
		<tr>
			<td>Anti PARP1 (F1-23)</td>
			<td>Homemade</td>
			<td></td>
			<td>1:2500</td>
		</tr>
		<tr>
			<td>Anti PCNA (SY12-07)</td>
			<td>Novus</td>
			<td>NBP2-67390</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Anti-Alpha tubulin (DM1A)</td>
			<td>Abcam</td>
			<td>Ab7291</td>
			<td>1:100000</td>
		</tr>
		<tr>
			<td>anti-BrdU</td>
			<td>GE Healthcare</td>
			<td>RPN202</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Benchtop X-ray Irradiator</td>
			<td>Cell Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BMN673</td>
			<td>MedChem Express</td>
			<td>HY-16106</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine (BrdU)</td>
			<td>Sigma</td>
			<td>B5002</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell profiler</td>
			<td>Broad Institute</td>
			<td>V 3.19</td>
			<td>https://cellprofiler.org/</td>
		</tr>
		<tr>
			<td>Curwood Parafilm M Laboratory Wrapping Film 4in / 250 ft</td>
			<td>Fisher scientific</td>
			<td>13-374-12</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen Life Technology</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Fisher scientific</td>
			<td>10063542</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>Gibco</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Cover Glasses: Squares 22 x 22</td>
			<td>Fisher scientific</td>
			<td>12 541B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Leica</td>
			<td>DMI6000B</td>
			<td>63x immersion objective</td>
		</tr>
		<tr>
			<td>HeLa</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HERACELL 160I CO2 INCUBATOR CU 1-21 TC 120V</td>
			<td>VWR</td>
			<td>51030408</td>
			<td>37% CO2</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>BioShop Canada</td>
			<td>MAG520.500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BioShop Canada</td>
			<td>SOD002.10</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Wisent Bio Products</td>
			<td>311-010-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 16%</td>
			<td>Cedarlane Labs</td>
			<td>15710-S(EM)</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>P6757-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Invitrogen Life Technology</td>
			<td>P-36930</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAiMAX</td>
			<td>Invitrogen</td>
			<td>13778-075</td>
			<td></td>
		</tr>
		<tr>
			<td>siPARPi</td>
			<td>Dharmacon</td>
			<td></td>
			<td>AAG AUA GAG CGU GAA GGC GAA dTdT</td>
		</tr>
		<tr>
			<td>siRNA control</td>
			<td>Dharmacon</td>
			<td></td>
			<td>UUCGAACGUGUCACGUCAA</td>
		</tr>
		<tr>
			<td>Sodium Deoxycholcate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BioShop Canada</td>
			<td>SUC507.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>BioShop Canada</td>
			<td>TRS001.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Trition X-100</td>
			<td>Millipore Sigma</td>
			<td>T8787-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Fisher scientific</td>
			<td>BP337500</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of global dna double-strand end resection using brdu-dna labeling coupled with cell cycle discrimination imaging

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). 
Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2&#8242;-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.

In this protocol, the bromodeoxyuridine (BrdU)-based assay was used to quantitatively measure the resection response of HeLa cells to irradiation-induced damage. The generated ssDNA tracks are visualized as distinct foci after immunofluorescence staining (Figure 1A). The identified foci were then quantified and expressed as the total integrated intensity of the BrdU staining in the nuclei (Figure 1B, Supplemental Figure S1, Supplemental Figure S2,</stron...

Watch this Scientific Journal Video about Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging at JoVE.com

Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging

In the present protocol, we demonstrate how to visualize DNA double-strand end resection during S/G2 phase of the cell cycle using an immunofluorescence-based method.

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs ...

This protocol allows for in cellulo quantification of DNA and resection following DNA damage. This technique's main advantage is the cell cycle marking which restricts the cells to S and G2 phase, ensuring the bromodeoxyuridine signal results from DNA and resection. Demonstrating the procedure will be Jean-Yves Masson, a principal investigator, and Sofiane Y.Mersaoui, a post-doctoral researcher.Working under a sterile culture hood, place a single coverslip in each well of a six-well plate for as many conditions needed. Plate approximately 150, 000 HeLa cells for transfection or drug treatment. On day three, following the overnight incubation with BrdU at a final concentration of 10 micromolar, irradiate the plates with a total dose of five gray of x-ray irradiation.After irradiation, return the plates for incubation at 37 degrees Celsius in a 5%carbon oxide humidified incubator for three hours. For pre-extraction, aspirate the medium and carefully wash the cells twice with PBS. After removing PBS, add two milliliters of pre-extraction buffer A and incubate the cells at four degrees Celsius on ice for 10 minutes.After 10 minutes, aspirate buffer A, add two milliliters of cytoskeleton stripping buffer B, and repeat the incubation. Then carefully aspirate buffer B and wash the cells with PBS. After aspirating PBS, fix the cells by adding two milliliters of 4%of paraformaldehyde under the chemical hood.Following removal of the paraformaldehyde and washes with PBS, cover the coverslips with 100%cold methanol for incubation at minus 20 degrees Celsius for five minutes and wash the coverslips twice with PBS. For permeabilization, incubate the cells with two milliliters of PBS containing 0.5%Tritan X-100 at room temperature. After 15 minutes, wash the coverslips three times with two milliliters of PBS.For immunostaining, add two milliliters of blocking buffer to each well. After one hour of incubation, add 100 microliters of primary antibody solution to each coverslip. Use tweezers to cover the coverslips with square parafilm and carefully position the parafilm to not create bubbles.Then cover the plate in aluminum foil and incubate the primary antibody overnight at four degrees Celsius in a humidified chamber. On the following day, remove the parafilm squares and wash the coverslips three times with two milliliters of PBS. After the wash, add 100 microliters of secondary antibody solution on each coverslip and cover the coverslips with a square of parafilm as demonstrated.Incubate the secondary antibody at room temperature for one hour and protect the plate from light using foil. Then wash the coverslips three times with two milliliters of 1X PBS. For nuclear staining, add two milliliters of DAPI solution to each coverslip and wash the coverslips with PBS.Add 10 to 20 microliters of IF-specific mounting medium on microscope slides. Use the needle or fine tweezers to lift the coverslip from the bottom of the well. Carefully blot off excess liquid by tapping one edge gently on a paper towel and mount the coverslips on microscope slides.For image acquisition and analysis, import these files into CellProfiler and analyze them using the speckle counting pipeline. Identify the primary object to identify the foci using the settings described in the text manuscript and measure the intensity. Representative images of 5-bromo-2-prime-deoxyuridine foci formation and proliferating cell nuclear antigen staining following irradiation are shown here.The generated single-stranded DNA tracks were visualized as distinct foci. The identified foci were then quantified and expressed as the total integrated intensity of the bromodeoxyuridine staining in the nuclei. Co-staining showed differentiation between the short range resection due to non-homologous end joining and the long range resection of homologous recombination using an anti-PCNA antibody to identify cells going through the S phase.PCNA is prominent in the nucleus and reaches maximal expression during the S phase of the cell cycle with quite an intense staining. The result in values are then plotted as a scatterplot to discriminate between PCNA positive and PCNA negative nuclei. The PCNA negative results are removed from the dataset to allow for BrdU intensity-based analysis because of the low BrdU signal regardless of the condition.The PCNA negative nuclei harbor a basal integrated intensity of BrdU foci of 900 arbitrary units and this value reaches 1, 800 AU in PCNA positive nuclei. In the siRNA control condition, the integrated intensity of BrdU foci per nucleus is approximately 1, 500 AU in the PCNA positive nuclei. After an efficient knockdown of PARP-1 using an siRNA, a significant increase in the intensity of the BrdU foci to approximately 1, 900 AU was observed.This technique can provide key insights into the first stage of homologous recombination, thus helping to identify if a protein is involved in the first step of the repair pathway. The incubation time for the pre-extraction buffers must be strictly kept. Over-exposure to these buffers will result in excessive cell detachment and increased background signal.

Research

JoVE Journal

Biology

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

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 ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy

Bdellovibrio bacteriovorus is a small gram-negative, obligate predatory bacterium that kills other gram-negative bacteria, including harmful pathogens. ...