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 possible to do a forward transfection several hours after plating to allow for adherence. A reverse transfection is accomplished by adding the transfection mix to the coverslip before the cells are added; thus, transfection begins before the cells start adhering. A forward transfection, by contrast, is the addition of the transfection mix post-adherence to the surface usually on the following day. However, it is possible to do this on the same day, provided enough time is given for the cells to adhere.</li>
		<li>For this method, use 4 wells: Well 1: siRNA control (termed siCTRL); Well 2: siRNA against PARP-1 (siPARP-1); Well 3: untreated; Well 4: Cells to be treated with 5 &#181;M BMN673 1 h prior to irradiation. 
		NOTE: In this protocol, all the tests were conducted under irradiated conditions (see section 1.3).</li>
		<li>Incubate the cells at 37 &#176;C in a 5% CO2 humidified incubator overnight, although the transfection protocol allows for up to 3 days of incubation. The incubation time prior to BrdU treatment will depend on the siRNA transfection efficiency. If there is no transfection, incubation for 16 h is sufficient to allow for adherence to the coverslip and some cell growth. 
		NOTE: Incubator conditions can be changed according to the cell line used.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Add BrdU at a final concentration of 10 &#181;M in the appropriate culturing media, and incubate for 16 h (one cell cycle). 
		NOTE: The BrdU solution is prepared in dimethylsulfoxide; the stock solution used is 10 mM and is stored in aliquots at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Day 3
	<ol>
		<li>Irradiate the plates with a total dose of 5 Gy of X-ray irradiation (vary the dose of irradiation depending on the irradiator type, for example, small animal irradiators vs. benchtop irradiators). See the Table of Materials for the brand and model of irradiator used.</li>
		<li>Return the plates to the incubator, and release the cells for 3 h.</li>
		<li>During the 3 h incubation period, prepare the two buffers, A and B, and 4% paraformaldehyde (PFA) in 1x phosphate-buffered saline (PBS). 
		NOTE: Buffers A and B and the sucrose should be prepared fresh the day of fixation, using fresh sucrose solution to prevent contamination.
		<ol>
			<li>Prepare buffer A (Pre-Extraction Buffer), according to the order (30 mL) described in Table 1. 
			NOTE: The 1M sucrose should be prepared the day of use to prevent contamination.</li>
			<li>Prepare buffer B (Cytoskeleton Stripping Buffer) according to the order described in Table 1 (30 mL). 
			&#8203;NOTE: Buffer B must be prepared in the cited order to prevent precipitation of Tween-20 and sodium deoxycholate solution.</li>
			<li>Prepare 4% PFA, 2 mL per condition (10 mL), under a chemical hood (Table 1).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Pre-extraction and fixation
NOTE: All pre-extraction and&#160;fixation are performed with the coverslips remaining in the tissue culture plate on ice or at 4 &#176;C; the coverslip is only lifted in the last step of mounting (see discussion).
<ol>
	<li>Pre-extraction
	<ol>
		<li>Aspirate the medium, carefully wash the cells twice with 1x PBS, and remove the PBS. Add 2 mL of Pre-extraction Buffer A and immediately incubate at 4 &#176;C for 10 min. 
		NOTE: It is important that incubation time in buffer A is not extended. Extended incubation in the pre-extraction buffers will result in an increased number of detached cells from the coverslip. Alternatively, instead of incubation at 4&#176;C, incubation can be done on ice.</li>
		<li>Remove Buffer A through aspiration. 
		NOTE: Do not wash the coverslips after removing buffer A. Proceed to the next step.</li>
		<li>Add 2 mL of Cytoskeleton Stripping Buffer B and immediately incubate at 4 &#176;C for 10 min. Carefully aspirate Buffer B, and carefully wash the cells once with 1x PBS. Carefully aspirate the 1x PBS.</li>
	</ol>
	</li>
	<li>Fixation
	<ol>
		<li>Fix the cells by adding 2 mL of 4% PFA under the chemical hood. 
		NOTE: PFA is toxic and must be manipulated under a chemical hood.</li>
		<li>Incubate the cells at room temperature for 20 min. Wash the coverslips twice with 1x PBS, and aspirate the excess.</li>
		<li>Cover the coverslips with 100% cold methanol, and incubate the coverslips at -20 &#176;C for 5 min. Wash twice with 1x PBS. 
		&#8203;NOTE: The protocol can be paused here. The coverslips can be stored at 4 &#176;C in 1x PBS and the plate wrapped in aluminum foil if necessary. The cells should not be kept more than 5 days before continuing the IF protocol.</li>
	</ol>
	</li>
</ol>
3. Permeabilization
<ol>
	<li>Incubate the cells with 2 mL of 1x PBS containing 0.5% Triton X-100 at room temperature for 15 min. Wash the coverslips three times with 2 mL of 1x PBS.</li>
</ol>
4. Immunostaining
<ol>
	<li>Blocking step
	<ol>
		<li>Prepare enough fresh blocking buffer (3% BSA in 1x PBS) to use 2 mL per coverslip. 
		&#8203;NOTE: Prepare an extra 5mL of blocking buffer to make the antibody solutions.</li>
		<li>Add 2 mL of blocking buffer to each well and incubate at room temperature for 1 h.</li>
	</ol>
	</li>
	<li>Primary antibody incubation
	<ol>
		<li>Prepare the primary antibody solution in fresh blocking buffer: BrdU RPN202 1:1000 and proliferating cell nuclear antigen (PCNA) 1:500, 100 &#181;L per coverslip. 
		NOTE: To preserve the antibody, a smaller volume can be used, 75-100 &#181;L for a 22 x 22 mm coverslip, 50-75 &#181;L for an 18 x 18 mm coverslip.</li>
		<li>On each coverslip, add 100 &#181;L of primary antibody solution. Cover the coverslips with a square of parafilm using tweezers, and carefully position the parafilm to not create bubbles.</li>
		<li>Cover the plate in aluminum foil, and incubate the primary antibody overnight at 4 &#176;C in a humidified chamber.</li>
		<li>Remove the parafilm squares, and wash the coverslips three times with 2 mL of sterile 1x PBS.</li>
	</ol>
	</li>
	<li>Secondary antibody incubation
	<ol>
		<li>Prepare enough secondary antibody solution in fresh blocking buffer: anti-mouse 488 fluorescent secondary A11011 (for BrdU) dilution 1:800 and anti-rabbit 568 fluorescent secondary A11011 (for PCNA) dilution 1:800. 
		NOTE: The colors used can be changed to suit the experimental needs. The specific brand used can be found in the Table of Materials.</li>
		<li>Add on each coverslip 100 &#181;L of secondary antibody solution, cover the coverslips with a square of parafilm using tweezers, and carefully position the parafilm without bubbles.</li>
		<li>Incubate the secondary antibody at room temperature for 1 h with the plate covered in aluminum foil.</li>
		<li>Wash the coverslips three times with 2 mL of 1x PBS.</li>
	</ol>
	</li>
	<li>Nuclear staining
	<ol>
		<li>Prepare a volume of 2 mL per coverslip of 1x PBS containing 4&#39;,6-diamidino-2-phenylindole (DAPI) at a final concentration of 1 &#181;g/mL (1:1000).</li>
		<li>Add on each coverslip 2 mL of the DAPI solution. Incubate the coverslips at room temperature for 10 min with the plate covered in aluminum foil. Wash the coverslips twice with 1x PBS.</li>
		<li>Cover the coverslips with 1x PBS, and use either a needle or fine tweezers to lift the coverslip from the bottom of the well.Carefully blot the off the excess liquid by tapping one edge gently on a paper towel. 
		NOTE: Be careful not to drop the slide or allow the flat surface with the adherent cells to touch the paper.</li>
		<li>Mount the coverslips on slides using 10-20 &#181;L of IF-specific mounting media.</li>
	</ol>
	</li>
</ol>
5. Image acquisition and analysis
NOTE: Image acquisition can be done on several types of fluorescent microscopes. An epifluorescence microscope with a 63x oil objective was used; see the Table of Materials for the brand and model. Z-stacks are not required, although they may be of use depending on the cell line and level of mitochondrial staining.
<ol>
	<li>Image analysis
	<ol>
		<li>For each image, create multiple tiff files; merge all planes, but keep the color channels separate. Import these files into Cell Profiler (The Broad Institute https://cellprofiler.org/home) and analyze using the Speckle Counting pipeline (Supplemental Video 1).</li>
		<li>Upload the images (Supplemental Figure S1A).
		<ol>
			<li>To begin creating the project, load the speckle counting pipeline into the program and the images to be analyzed in the provided area.</li>
			<li>Use the NamesAndTypes module to assign a meaningful name to each image by which other modules will refer to it-C01: Representing BrdU Channel; C02: Representing PCNA Channel; C03: Representing DAPI Channel. 
			NOTE: These identifiers will depend on the microscope; there should be an identifier in the file name to distinguish between the channels.</li>
		</ol>
		</li>
		<li>Identify which file will be used to identify the nuclei and name it (change this name as required) (see Supplemental Figure S1B and Supplemental Figure S1C).
		<ol>
			<li>Select the diameter of the object between 50 and 300 pixel units, which is the acceptable size range for nuclei, but change the value to fit the nuclei in the image. 
			NOTE: It may be that 50 is too large a value and prevents smaller nuclei from being identified; likewise, 300 may allow for large groupings of nuclei to be counted as 1.</li>
			<li>Discard objects outside of the diameter range to ensure only those that match the criteria will be counted.</li>
			<li>Discard objects touching borders to remove cells that may only be partially in the field.</li>
			<li>Apply the threshold to fit the specific images. Note the following settings as an example. Change the thresholding correction factor based on how stringent the thresholding strategy will be. Other settings include threshold strategy: Global; thresholding method: Otsu; two class or three class thresholding: Two class; threshold smoothing scale: 1; threshold smoothing factor: 1; lower and upper bounds on threshold: 0.0 and 1.0; method to distinguish clumped objects: Shape; and method to draw dividing lines between clumped objects: Propagate. 
			NOTE: For each parameter, cell profiler provides complementary definitions and available possibility for the variable setting.</li>
		</ol>
		</li>
		<li>Identify the primary object to identify the foci (Supplemental Figure S2A).
		<ol>
			<li>Use the following settings: select the input image: Maskedgreen; named the primary object to be identified: BrdUFoci; typical diameter of the object: 1-15; discard the object outside the diameter range: Yes; discard the object touching the border of the image : No; threshold strategy: Adaptative; thresholding method: Otsu; two class or three class thresholding: Three class; threshold smoothing scale: 1; threshold smoothing factor: 3; and size of smoothing filter: 4.</li>
		</ol>
		</li>
		<li>Measure intensity (Supplemental Figure S2B).
		<ol>
			<li>Select the image to be measured: OrigGreen.</li>
			<li>Click on Add another image. Select the image to be measured: PCNA. Select objects to measure: Nuclei. 
			NOTE: This will be used to measure the BrdU and PCNA intensity-the final data that will be graphed.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 ...

assessment-global-dna-double-strand-end-resection-using-brdu-dna

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

3.8K Views.  Genome Stability Laboratory, CHU de Québec Research Center, HDQ Pavilion. 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.

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

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 imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



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 imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

Pomiar egzocytozy w Neurony Korzystanie FM etykietowania

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 medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

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 labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

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

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 two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

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

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining 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 quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

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 cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

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

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

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

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

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 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 ...

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 not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

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

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

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

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Pomiar egzocytozy w Neurony Korzystanie FM etykietowania

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

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

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

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

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

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

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring