This work was supported by the National Institute on Aging grant R03AG064525 to ASW. We would like to thank Dr. Jason A. Stewart for help in technique mentorship and the Hui Chen laboratory for providing the microscope used. We would also like to thank Fabio Martinon for the HeLa cell lines used in these experiments.

Details of the reagents and the equipment used are listed in the Table of Materials.
1. Preparation of materials
<ol>
	<li>Autoclave at least ten 22 mm x 22 mm square glass coverslips and forceps in advance of plating cells onto the coverslips.</li>
</ol>
2. Preparation of the solutions
<ol>
	<li>Supplemented media: Prepare 500 mL of Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 &#181;g/mL streptomycin.</li>
	<li>Antibody dilution buffer (ADB): Dissolve 2 g of bovine serum albumin (BSA) and 1 g of Fish gelatin in 80 mL of 1x phosphate-buffered saline (PBS). Bring to 100 mL with PBS once BSA and fish gelatin are dissolved.</li>
	<li>1x PBS-Tween (PBS-T): Prepare 500 mL of PBS and 0.5 mL of Tween. Place in a 37 &#176;C water bath until Tween can be easily mixed into the solution.</li>
</ol>
3. Growing the desired cells
NOTE: HeLa cells were grown in this protocol to confluency within a T75 flask.
<ol>
	<li>Spray six coverslips with 70% ethanol and allow them to air dry on a lint-free wipe. Place coverslips into a sterile 6-well plate using forceps, one coverslip per well.</li>
	<li>Add 2 mL of supplemented DMEM to each well in the 6-well plate.</li>
	<li>Aspirate media from the cells and wash the cells by adding 15 mL of PBS to the flask, gently swirling. Aspirate the PBS and add 2 mL of trypsin-EDTA (trypsin).
	<ol>
		<li>Coat the cells with trypsin and place the flask back into a 37 &#176;C and 5% CO2 humidified incubator for 2 min to allow cells to detach from the flask.</li>
	</ol>
	</li>
	<li>Add 8 mL of supplemented DMEM to the flask to collect the trypsinized cells. Pipette up and down a few times to separate clumping cells. Collect the cell suspension in the flask and place it into a 15 mL polystyrene tube.</li>
	<li>Count the cells using a hemocytometer.</li>
	<li>Add 500,000 cells, drop by drop, directly onto each of the coverslips in the 6-well plate. Allow cells to grow overnight at 37 &#176;C and 5% CO2 in a humidified incubator.</li>
</ol>
4. Cell fixation and permeabilization
<ol>
	<li>Prepare the 4% formaldehyde solution fresh before use: Add 2 mL of 37.5% formaldehyde solution into 18 mL of PBS. Mix thoroughly.</li>
	<li>Prepare the permeabilization solution fresh before use: Add 100 &#181;L of Triton-X into 20 mL of PBS. Allow to incubate in a 37 &#176;C water bath for a few minutes until the Triton-X dissolves into the solution. Mix thoroughly.</li>
	<li>Aspirate the media in the wells of the plate. Wash cells once with 2 mL of PBS. Aspirate all PBS from the wells.</li>
	<li>Add 2 mL of 4% formaldehyde solution to each well. Allow to sit for 10 min at room temperature (RT). Aspirate 4% formaldehyde completely from the wells.</li>
	<li>Wash cells 3 times with 2 mL of PBS. Remove PBS completely from the wells after each wash.</li>
	<li>Add 2 mL of permeabilization solution to each well. Allow to sit for 10 min at RT. Aspirate permeabilization solution completely.</li>
	<li>Wash cells 3 times with 2 mL of PBS. Aspirate PBS completely after the first two washes, leaving the PBS in the wells after the third wash. 
	NOTE: Cells can be stored in the last PBS wash at 4 &#176;C for a few weeks after this step, ensuring the plate is sealed with paraffin film to avoid liquid evaporation. If the procedure can be continued, this step can be skipped.</li>
</ol>
5. Immunofluorescence staining
<ol>
	<li>Remove all PBS from wells. Block cells by adding 2 mL of ADB to each well and incubate at RT, rocking for 30 min.</li>
	<li>Towards the end of the 30 min blocking time, mix the primary antibody dilution in ADB (1:1,000 dilution for both Lamin B1 and dsDNA antibodies, Table 1). 75 &#181;L is needed for each coverslip, therefore 600 &#181;L of ADB, 0.6 &#181;L of Lamin B primary antibody, and 0.6 &#181;L of dsDNA antibody should be mixed for six coverslips. Vortex thoroughly.</li>
	<li>Remove ADB from the wells. On a flat surface, tape down a piece of paraffin film that is large enough in area to lay down all the coverslips being processed.</li>
	<li>For each coverslip, add 75 &#181;L of primary antibody dilution onto the paraffin film, ensuring no bubbles are pipetted in the ADB. Then, place the coverslips cell side down onto the droplet of ADB using forceps.</li>
	<li>Cover the incubating coverslips with the well plate lid and allow to incubate for 1 h at RT.</li>
	<li>Remove the coverslips from the paraffin film using forceps and place the coverslips back into the 6-well plate, cell side facing upward.</li>
	<li>Wash the coverslips 3 times with 2 mL of PBS-T, rocking for 3-5 min each time. Remove all PBS-T from the wells after each wash.</li>
	<li>During the last wash, prepare the secondary antibody dilution (1:1,000 for both respective secondary antibodies, Table 1). 75 &#181;L is needed for each coverslip, therefore 600 &#181;L of ADB and 0.6 &#181;L of both secondary Alexa Fluor tagged antibodies should be mixed for six coverslips. Vortex thoroughly. Cover the secondary antibody dilution from light as much as possible.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Antibody</td>
			<td>Source</td>
			<td>Company</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td colspan="3">dsDNA Marker Antibody (HYB331-01)</td>
			<td>Mouse monoclonal</td>
			<td>Santa Cruz</td>
			<td>sc-58749</td>
		</tr>
		<tr>
			<td colspan="3">Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488</td>
			<td>Goat</td>
			<td>ThermoFisher&#160;</td>
			<td>A32723</td>
		</tr>
		<tr>
			<td colspan="3">Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 405</td>
			<td>Goat</td>
			<td>Invitrogen</td>
			<td>A31553</td>
		</tr>
		<tr>
			<td colspan="3">Lamin B1 Polyclonal Antibody</td>
			<td>Rabbit</td>
			<td>Proteintech</td>
			<td>12987-1-AP</td>
		</tr>
		<tr>
			<td colspan="3">Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594</td>
			<td>Goat</td>
			<td>Invitrogen</td>
			<td>A11037</td>
		</tr>
	</tbody>
</table>
Table 1: Antibodies. List of all antibodies used in this protocol.
<ol start="9">
	<li>Remove all PBS-T from the last wash. Incubate the coverslips in the secondary antibody dilution on paraffin film, as in step 5, for 30 min at RT. Protect from light for this step and all remaining steps. A small box can be used to shield light. Ensure to work quickly when needed to add or remove solutions from the wells.</li>
	<li>Remove coverslips from the paraffin film using forceps and place the coverslips back into the 6-well plate, cell side up.</li>
	<li>Wash the coverslips 3 times with 2 mL of PBS-T. Remove all PBS-T from the wells after each wash.</li>
	<li>Dehydrate the slides with an ethanol series (70%, 90%, 100%). Start by adding 2 mL of 70% ethanol to each well. Allow to sit for 1-2 min, and remove all ethanol from the wells. Repeat with the remaining ethanol percentages.</li>
	<li>Remove the coverslips from the wells using forceps and let them air-dry, protected from light, on a lint-free wipe.</li>
	<li>Mount the coverslip&#39;s cell side down onto glass microscope slides using 20 &#181;L of mounting medium per coverslip. Allow to dry overnight, protected from light.</li>
</ol>
6. Image acquisition
<ol>
	<li>Before imaging, coat the edges of coverslips with a thin layer of clear nail polish (any brand or type will suffice). Allow to air dry, protected from light. 
	NOTE: The instructions for using a microscope to image will vary, depending on the microscope used. The resolution of the camera on the microscope used here is 1280 x 960 pixels. Similar or better resolution will suffice.</li>
	<li>Before putting a slide on the microscope stage, the file-saving settings must be adjusted. Turn on the microscope and insert a USB drive. Adjust the save settings within the microscope settings and ensure that all channels (GFP and RFP) will be saved separately and that the images are going to the correct location on the USB drive. 
	NOTE: Create a folder on the USB drive and give each coverslip its own name for future reference.</li>
	<li>Prior to imaging, spray a lint-free wipe with 70% ethanol and gently clean off the back of the slides. Gently clean the front of the slides, ensuring very minimal pressure is exerted.</li>
	<li>Place a clean slide onto the microscope, coverslips facing downward. Cover with the light shield box.</li>
	<li>Using a 40x objective lens, use the appropriate channel (RFP for the lamin B1 antibody employed here) to locate the nuclei on the microscope screen. Adjust the light intensity to around 50% to start.</li>
	<li>To check for oversaturated areas, select the Color Off option and ensure no areas across the coverslip have a red signal in the black and white setting. If there is a lot of red signal, lower the light intensity until the minimal red signal is shown. 
	NOTE: A red signal indicates oversaturation. If the nuclei appear barely visible, increase the intensity by 10% increments until an appropriate intensity is achieved without oversaturation.</li>
	<li>Check all the other coverslips with the same intensity to ensure they are not oversaturated. Check all coverslips with the other channel using the same process as above (GFP for the dsDNA antibody used here).</li>
	<li>Record light intensities for each channel. Ensure all the light intensities for each channel are kept constant throughout the imaging process.</li>
	<li>Image each coverslip individually. Start off with the first channel, focus the nuclei, and capture the image.</li>
	<li>Carefully switch channels and bring the cells into focus again. When focused, capture the image.</li>
	<li>After capturing images in the necessary channels (GFP and RFP), select Overlay to observe the merged image and ensure the slide did not move when taking the image. If the channels do not overlap, redo the necessary channel until the images line up.</li>
	<li>Save the image by clicking on the flash drive icon. Move to a different place on the coverslip to continue imaging. 
	NOTE: Ensure that a different area of the coverslip is imaged each time to avoid capturing the same region repeatedly. It is recommended to start imaging at the upper left-hand corner of the coverslip and move in an &#34;S&#34; pattern to ensure complete coverage without duplication.</li>
</ol>
7. Data analysis
<ol>
	<li>Data obtained with this method is visual. Examine the images for signs of DNA leakage or nuclear blebbing. Examples of blebbing and leakage are shown in the representative results section.</li>
</ol>

<ol>
	<li>Navarro, C. L., 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=New+ZMPSTE24+(FACE1)+mutations+in+patients+affected+with+restrictive+dermopathy+or+related+progeroid+syndromes+and+mutation+update.">New ZMPSTE24 (FACE1) mutations in patients affected with restrictive dermopathy or related progeroid syndromes and mutation update.</a> Eur J Hum Genet. 22 (8), 1002-1011 (2014).</li><li>Dittmer, T. A., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lamin+protein+family.">The lamin protein family.</a> Genome Biol. 12 (222), 1-16 (2011).</li><li>Gruenbaum, Y., Margalit, A., Goldman, R. D., Shumaker, D. K., Wilson, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+lamina+comes+of+age.">The nuclear lamina comes of age.</a> Nat Rev Mol Cell Biol. 6, 21-31 (2005).</li><li>Navarro, C. L., 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=Lamin+A+and+ZMPSTE24+(FACE-1)+defects+cause+nuclear+disorganization+and+identify+restrictive+dermopathy+as+a+lethal+neonatal+laminopathy.">Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganization and identify restrictive dermopathy as a lethal neonatal laminopathy.</a> Hum Mol Genet. 13 (20), 2493-2503 (2004).</li><li>Barrowman, J., Wiley, P. A., Hudon-Miller, S. E., Hrycyna, C. A., Michaelis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+ZMPSTE24+disease+mutations:+Residual+proteolytic+activity+correlates+with+disease+severity.">Human ZMPSTE24 disease mutations: Residual proteolytic activity correlates with disease severity.</a> Hum Mol Genet. 21 (18), 4084-4093 (2012).</li><li>Richards, S., Muter, J., Ritchie, P., Lattanzi, G., Hutchison, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+accumulation+of+un-repairable+DNA+damage+in+laminopathy+progeria+fibroblasts+is+caused+by+ROS+generation+and+is+prevented+by+treatment+with+N-acetyl+cysteine.">The accumulation of un-repairable DNA damage in laminopathy progeria fibroblasts is caused by ROS generation and is prevented by treatment with N-acetyl cysteine.</a> Hum Mol Genet. 20 (20), 3997-4004 (2011).</li><li>Davies, B. S., Fong, L. G., Yang, S. H., Coffinier, C., Young, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+post-translational+processing+of+prelamin+A+and+disease.">The post-translational processing of prelamin A and disease.</a> Annu Rev Genomics Hum Genet. 10, 153-174 (2009).</li><li>Barrowman, J., Hamblet, C., George, C. M., Michaelis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+prelamin+A+biogenesis+reveals+the+nucleus+to+be+a+CaaX+processing+compartment.">Analysis of prelamin A biogenesis reveals the nucleus to be a CaaX processing compartment.</a> Mol Biol Cell. 19, 5398-5408 (2008).</li><li>Young, S., Meta, M., Yang, S. H., Fong, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prelamin+A+farnesylation+and+progeroid+syndromes.">Prelamin A farnesylation and progeroid syndromes.</a> J Biol Chem. 281 (52), 39741-39745 (2006).</li><li>Houthaeve, G., Robijns, J., Braeckmans, K., De Vos, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bypassing+border+control:+Nuclear+envelope+rupture+in+disease.">Bypassing border control: Nuclear envelope rupture in disease.</a> Physiol. 33 (1), 39-49 (2017).</li><li>Schreiber, K. H., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+lamins+go+bad:+Nuclear+structure+and+disease.">When lamins go bad: Nuclear structure and disease.</a> Cell. 152 (6), 1365-1375 (2013).</li><li>Fong, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterozygosity+for+Lmna+deficiency+eliminates+the+progeria-like+phenotypes+in+Zmpste24-deficient+mice.">Heterozygosity for Lmna deficiency eliminates the progeria-like phenotypes in Zmpste24-deficient mice.</a> Proc Natl Acad Sci USA. 101 (52), 18111-18116 (2004).</li><li>Pendas, A. M., 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=Defective+prelamin+A+processing+and+muscular+and+adipocyte+alterations+in+Zmpste24+metalloproteinase-deficient+mice.">Defective prelamin A processing and muscular and adipocyte alterations in Zmpste24 metalloproteinase-deficient mice.</a> Nat Genet. 31, 94-99 (2002).</li><li>Goldman, R. D., 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=Accumulation+of+mutant+lamin+A+causes+progressive+changes+in+nuclear+architecture+in+Hutchinson-Gilford+progeria+syndrome.">Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome.</a> Proc Natl Acad Sci USA. 101, 8963-8968 (2004).</li><li>Eriksson, M., 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=Recurrent+de+novo+point+mutations+in+lamin+A+cause+Hutchinson&#8211;Gilford+progeria+syndrome.">Recurrent de novo point mutations in lamin A cause Hutchinson&#8211;Gilford progeria syndrome.</a> Nature. 423, 293-298 (2003).</li><li>Young, S. G., Fong, L. G., Michaelis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+Lipid+post-translational+modifications.+Prelamin+A,+Zmpste24,+misshapen+cell+nuclei,+and+progeria&#8212;new+evidence+suggesting+that+protein+farnesylation+could+be+important+for+disease+pathogenesis.">Thematic review series: Lipid post-translational modifications. Prelamin A, Zmpste24, misshapen cell nuclei, and progeria&#8212;new evidence suggesting that protein farnesylation could be important for disease pathogenesis.</a> J Lipid Res. 46 (12), 2531-2558 (2005).</li><li>Worman, H. J., Michaelis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prelamin+A+and+ZMPSTE24+in+premature+and+physiological+aging.">Prelamin A and ZMPSTE24 in premature and physiological aging.</a> Nucleus. 14 (1), 1-17 (2023).</li><li>Pande, S., Ghosh, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+proteostasis+imbalance+in+laminopathy-associated+premature+aging+diseases.">Nuclear proteostasis imbalance in laminopathy-associated premature aging diseases.</a> FASEB J. 37 (8), 1025-1035 (2023).</li><li>Odell, I. D., Cook, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+techniques.">Immunofluorescence techniques.</a> J Invest Dermatol. 133 (4), 1-12 (2013).</li><li>Kumar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+and+enzyme+immunomicroscopy+methods.">Immunofluorescence and enzyme immunomicroscopy methods.</a> J Immunoassay Immunochem. 21 (2-3), 235-253 (2000).</li><li>Tjaden, A., Giessmann, R. T., Knapp, S., Schroder, M., Muller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-content+live-cell+multiplex+screen+for+chemogenomic+compound+annotation+based+on+nuclear+morphology.">High-content live-cell multiplex screen for chemogenomic compound annotation based on nuclear morphology.</a> STAR Protoc. 3 (101791), 1-15 (2022).</li><li>Gunn, A. L., Yashchenko, A. I., Dubrulle, J., Johnson, J., Hatch, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-content+screen+reveals+new+regulators+of+nuclear+membrane+stability.">A high-content screen reveals new regulators of nuclear membrane stability.</a> Sci Rep. 14, 6013 (2024).</li><li>Yu, L., Liu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytosolic+DNA+sensing+by+cGAS:+Regulation,+function,+and+human+diseases.">Cytosolic DNA sensing by cGAS: Regulation, function, and human diseases.</a> Nat Rev Mol Cell Biol. 6 (170), 1-14 (2021).</li><li>Odinammadu, K. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+farnesyl+transferase+inhibitor+(FTI)+lonafarnib+improves+nuclear+morphology+in+ZMPSTE24-deficient+fibroblasts+from+patients+with+the+progeroid+disorder+MAD-B.">The farnesyl transferase inhibitor (FTI) lonafarnib improves nuclear morphology in ZMPSTE24-deficient fibroblasts from patients with the progeroid disorder MAD-B.</a> Nucleus. 14 (1), 1-14 (2023).</li></ol>

The authors have nothing to disclose.

The protocol presented contains several critical steps, the most important being the treatment of the fixed cells with primary and secondary antibodies. Ensuring the use of a good quality primary antibody against the target of interest with a correct corresponding secondary antibody with fluorophores within the range of the microscope used will yield optimal results19. The quality of the antibody can greatly impact the results of this technique, as faulty antibodies can interact with nonspecific targets or not recognize the target of interest19,20. Additionally, if there is little to no signal, the dilution of the primary and secondary antibodies should be adjusted to ensure enough signal is detected over the background, taking care not to oversaturate the signal19,20. Optimal blocking conditions can minimize these imaging artifacts20. For IF, the precise antibody dilutions will likely have to be adjusted for each individual system to ensure success, but a successful dilution tends to be within the manufacturer&#39;s suggested range. Along with dilutions, the timing of the protocol should be followed carefully. Overtreatment or undertreatment of the cells with solutions and antibodies can create imaging artifacts or damage the cells19. Lastly, it is important that once the coverslips are treated with the secondary antibody, they should be shielded from light for the remainder of the protocol. If exposed to too much light, the samples could be photobleached and may not fluoresce properly when imaged, or the coverslips will not be stored for a long period20. To prevent photobleaching, it is vital to protect IF slides from light when storing the slides.&#160; We also recommend using an antifade mounting medium to preserve signal19.
Though the method described is useful for detecting nuclear blebbing and DNA leakage into the cytosol, it does have limitations in that it is not performed on live cells. Since this technique is performed on fixed cells, it cannot be determined which cells are still viable after leakage of DNA into the cytosol occurs. Incorporating live cell imaging along with this protocol would allow users to gain insight into the cell viability post leakage, along with visualizing nuclear blebbing in real time21,22. Secondly, as mentioned, this technique may require a number of adjustments to various parts of the protocol to ensure success, particularly with antibody dilutions and the number of cells plated. Lastly, this technique, in comparison to existing methods, is an initial screening to determine if laminopathy cells exhibit these phenotypes. Detection of nuclear blebbing and DNA leakage with this protocol will provide preliminary data to determine if other methods, such as live cell imaging, are warranted21,22. Additionally, this protocol could be used alongside other protocols that employ antibodies to detect other factors, like cGAS, which responds to the presence of cytosolic DNA, to confirm and expand findings23.
Overall, IF is a vital technique in the field of investigating laminopathy disorders and will continue to be used to contribute to our knowledge of nuclear structure and function. Observations of nuclear structure contribute to our understanding of the characteristics of laminopathies and other disorders of the nucleus. Such observations can also aid in gauging the efficacy of potential treatments for these disorders. The IF procedure described here can be used to screen compounds to assess their ability to improve the nuclear structure or leakage seen in laminopathies, and such data may serve as a sentinel of a compound&#39;s potential therapeutic usefulness24.

The nuclear lamina is a network of filaments that underlies the nuclear membrane and is made up of proteins called lamins. The nuclear lamina plays essential roles in nuclear architecture, positioning of nuclear pores, gene expression regulation, chromatin organization, DNA replication, and DNA repair1,2,3. Mutations in genes that play a role in the expression of the lamin proteins lead to genetic disorders called laminopathies3.
Restrictive dermopathy (RD) is a severe laminopathy disorder predominantly caused by compound heterozygous mutations that create premature termination codons in the ZMPSTE24 gene4, leading to an absence of the metalloprotease ZMPSTE24. This laminopathy is characterized by intrauterine growth retardation, tight, rigid skin, small-sized mouth, thin hair, and bone mineralization defects1. RD patients usually do not live past their first week of life due to pulmonary insufficiency1. Two other laminopathy disorders known as atypical Hutchinson-Gilford Progeria Syndrome (AT-HGPS) and Mandibuloacral dysplasia type B (MAD-B) involve reduced ZMPSTE24 expression and are associated with reduced lifespan and show similarities to premature aging disorders5. The ZMPSTE24 protease that is impacted in these laminopathies is vital in the post-translational modification of lamin A, which is a critical component of the nuclear lamina. ZMPSTE24 deficiency results in the accumulation of an incompletely processed farnesylated form of lamin A, known as prelamin A2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67719/67719fig1.jpg" /> 
Figure 1: Lamin A processing in normal vs. RD cells. Lamin A processing pathway in normal cells (left) and altered lamin A processing in RD, or ZMPSTE deficient cells (right). Farnesyl (Fa) and methyl (Me) groups are indicated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67719/67719fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As seen in Figure 1, in normal lamin A processing, a farnesyl (lipid) group is attached to a cysteine residue near the C-terminus, followed by proteolytic cleavage of three C-terminal amino acids3,6. The cysteine residue is then methylated. These two modifications allow prelamin A to be targeted to the inner nuclear membrane2. ZMPSTE24 then carries out a cleavage reaction whereby the last 15 C-terminal amino acids, along with the farnesyl and methyl groups, are removed to produce mature Lamin A protein, which is delivered to the nuclear lamina6,7,8. In RD, AT-HGPS, and MAD-B, the final step of processing does not occur effectively since the ZMPSTE24 metalloproteinase is lacking or not fully functional8,9. This results in the accumulation of prelamin A, which remains permanently farnesylated and methylated9. These latter groups cause the prelamin A to stick to the inner nuclear membrane instead of localizing to the nuclear lamina, where mature lamin A should ultimately reside, as seen in Figure 1. Thus, ZMPSTE deficiency has profound effects on a variety of nuclear functions, along with nuclear structure10,11,12,13,14,15. These alterations to nuclear structure may include nuclear blebbing and even nuclear rupturing which can result in the leakage of DNA into the cytosol as well as ingress of cytosol into the nucleus. Aberrantly shaped nuclei are indeed a hallmark of laminopathy disorders, along with numerous other phenotypes that are caused by defective prelamin A processing16,17. Defects in the nuclear lamina lead to a multitude of detrimental impacts, including the mislocalization of nuclear protein quality control and DNA repair proteins in the nucleoplasm, which, in turn, results in numerous defects in nuclear function18. It is important to have a simple technique that can help to identify and monitor these hallmarks of laminopathies to facilitate research into the development of therapeutic approaches aimed at ameliorating laminopathy phenotypes. For mice with laminopathies, it has been shown that the elimination of nuclear blebbing correlates with the generalized elimination of laminopathy phenotypes16. A technique that allows monitoring of nuclear integrity in human cells could enhance the study of potential treatments for eliminating or significantly improving disease phenotypes.
Indirect immunofluorescence (IF) is a sensitive and widely used technique that uses both a primary unlabeled antibody and a fluorophore-labeled secondary antibody that recognizes the primary antibody to detect a target of interest19. IF methods can provide a powerful means for visualizing specific intracellular components and structures. It is also possible that more than one secondary antibody molecule may interact with the primary antibody, resulting in amplification of the signal19. Indirect IF is a versatile technique that also allows the detection of multiple primary antibodies with a relatively small set of secondary antibodies since secondary antibodies are raised against the Fc domain of the primary antibody, which is conserved within species19.
This article describes an indirect&#160;(IF) method to assess nuclear blebbing and DNA leakage in cells deficient in ZMPSTE24, using antibodies against double-stranded DNA (dsDNA) and lamin B1 to detect DNA and the nuclear lamina, respectively. To demonstrate the utility of this approach, the procedure was applied to a HeLa cell line with knocked-out ZMPSTE24 expression, as well as a HeLa cell line that expresses ZMPSTE24, and the outcomes for the two cell lines were compared.

<table><tbody><tr><td>22 mm Square Glass Coverslips</td><td>Propper Manufacturing Company</td><td>M8710</td><td>Any size or shape can be used as long as they can be fixed to a standard microscope slide.&amp;nbsp;</td></tr><tr><td>36.5% Formaldehyde</td><td>Sigma Aldrich</td><td>F8775</td><td>Fixation Reagent.&amp;nbsp;</td></tr><tr><td>75 cm 2 Flasks</td><td>Corning</td><td>430725U</td><td>For cell culture.&amp;nbsp;</td></tr><tr><td>Bovine Serum Albinum (BSA)</td><td>Fisher Scientific</td><td>9048-46-8</td><td>Antibody dilution buffer.&amp;nbsp;</td></tr><tr><td>DMEM with 1 g/L glucose, L-glutamine &amp;amp; sodium pyruvate</td><td>Corning</td><td>10-014-CV</td><td>For cell culture.&amp;nbsp;</td></tr><tr><td>Ethanol (200 Proof)</td><td>Decon Laboratories</td><td>2701</td><td>For dehydration of samples before mounting, diluted to make multiple concentrations.&amp;nbsp;</td></tr><tr><td>EVOS FL Digital Inverted Fluorescence Microscope</td><td>Fisher Scientific</td><td>12-563-460</td><td>Imaging.&amp;nbsp;</td></tr><tr><td>Fish Gelatin</td><td>Sigma Aldrich</td><td>G7041</td><td>Antibody dilution buffer.&amp;nbsp;</td></tr><tr><td>Fluoromount-G</td><td>Southern Biotech</td><td>0100-01</td><td>Mounting medium to prevent photobleaching.&amp;nbsp;</td></tr><tr><td>Parafilm (4 in)</td><td>Fisher Scientific</td><td>13-374-12</td><td>Any size can be substituted, as long as the coverslips being used can fit.&amp;nbsp;</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15-140-122</td><td>For cell culture.&amp;nbsp;</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>N/A</td><td>N/A</td><td>Made and sterilized in the lab for tissue culture and solutions.</td></tr><tr><td>Premium Fetal Bovine Serum (FBS)</td><td>Atlanta Biologicals</td><td>S11150</td><td>For cell culture.&amp;nbsp;</td></tr><tr><td>Superfrost Premium Microscope Slides</td><td>Fisher Scientific</td><td>12-544-7</td><td>Any standard microscope slides can be used.&amp;nbsp;</td></tr><tr><td>Tissue Culture Treated 6-well Flat Bottom Plates</td><td>Falcon</td><td>353046</td><td>For cell culture.&amp;nbsp;</td></tr><tr><td>TritonX-100</td><td>Thermofisher Scientific</td><td>A16046.AP</td><td>For washing.</td></tr><tr><td>Trypsin-EDTA (0.5%)</td><td>Gibco</td><td>15-400-054</td><td>For cell culture.&amp;nbsp;</td></tr><tr><td>Tween 20</td><td>Fisher Scientific</td><td>9005-64-5</td><td>Cell Permeation reagent.&amp;nbsp;</td></tr></tbody></table>

detection of nuclear blebbing and dna leakage in mammalian cells by immunofluorescence

The nuclear lamina is a network of filaments underlying the nuclear membrane, composed of lamins and lamin-associated proteins. It plays critical roles in nuclear architecture, nuclear pore positioning, gene expression regulation, chromatin organization, DNA replication, and DNA repair. Mutations in genes involved in the expression or post-translational processing of lamin proteins result in genetic disorders known as laminopathies. Specifically, mutations in the LMNA or ZMPSTE24 genes can lead to the accumulation of incompletely processed forms of lamin A that retain farnesyl and methyl groups, which are absent in fully processed lamin A. These incompletely processed lamin A proteins localize to the inner nuclear membrane instead of the nuclear lamina, where mature lamin A resides. Mislocalized lamin proteins profoundly disrupt nuclear function and structure, often resulting in nuclear blebbing. In severe cases, nuclear rupture can occur, causing a loss of compartmentalization and leakage of genomic DNA into the cytosol. Abnormal nuclear structure and compartmentalization loss can be identified through indirect immunofluorescence (IF) on fixed cells. This study outlines such a method, employing specific antibodies against a lamin protein and double-stranded DNA (dsDNA) to simultaneously visualize the nuclear envelope and DNA. This approach enables a rapid assessment of nuclear structural integrity and the potential leakage of nuclear DNA into the cytosol.

This study introduces an IF method for visualizing the nuclear lamina in conjunction with double-stranded DNA (dsDNA). Once IF is performed, the images captured can be examined for signs of nuclear blebbing and DNA leakage.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67719/67719fig2.jpg" /> 
Figure 2: Nuclear blebbing in HeLa CT and ZMPSTE24 KO cells. Representative images of both cell lines used to determine the frequency of blebbed nuclei. (A) The color red (Alexa Fluor 594 conjugated secondary) was used to visualize the nuclear lamina (Lamin B1) of the cells. Examples of nuclei displaying no blebbing or blebbing of various severities are shown, with blebs indicated by white arrows. The scale bar represents 100 &#181;m (40x magnification). (B) Quantification of nuclear blebbing within the HeLa control (CT) and HeLa ZMPSTE24 KO cells, pooled from two biological replicates. Cell lines were scored double-blind for the presence of no blebbing or one or more blebs present. The difference between these groups was found to be highly significant by Chi-Square analysis using Excel (p &#60; 0.00001). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67719/67719fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Line</td>
			<td>Expt. No.</td>
			<td>Blebbed</td>
			<td>Not Blebbed</td>
			<td>Percent of Nuclei Blebbed</td>
		</tr>
		<tr>
			<td rowspan="3">HeLa Control</td>
			<td>1</td>
			<td>47</td>
			<td>353</td>
			<td>11.8</td>
		</tr>
		<tr>
			<td>2</td>
			<td>96</td>
			<td>304</td>
			<td>24</td>
		</tr>
		<tr>
			<td colspan="3">Average</td>
			<td>17.9</td>
		</tr>
		<tr>
			<td colspan="5"></td>
		</tr>
		<tr>
			<td rowspan="3">HeLa ZMPSTE24 KO</td>
			<td>1</td>
			<td>225</td>
			<td>175</td>
			<td>56.2</td>
		</tr>
		<tr>
			<td>2</td>
			<td>199</td>
			<td>201</td>
			<td>49.8</td>
		</tr>
		<tr>
			<td colspan="3">Average</td>
			<td>53</td>
		</tr>
	</tbody>
</table>
Table 2: Replicates of nuclear blebbing quantification in HeLa CT and ZMPSTE24 KO cells. Individual biological replicates performed to determine the pooled data for Figure 2B.
Nuclear blebbing can be examined by using a primary antibody against Lamin B1 (Table 1). Figure 2A shows images displaying positive and negative examples of blebbing. Cells are &#34;negative&#34; for blebbing if the nuclei are smooth, with a circular or oval shape. Cells are &#34;positive&#34; for blebbing if the nuclei are abnormally shaped or have any projections protruding outwards, indicated by arrows (Figure 2A). Blebbing was found to be prevalent in the ZMPSTE24 KO cells, with about 50% of the cells scored containing one or more blebs (Figure 2B). For the control (CT) cells, only about 17% of the cells scored were found to display blebbed nuclei. Table 2 shows the breakdown of each individual biological replicate that was performed to determine the percentage of blebbed nuclei.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67719/67719fig3.jpg" /> 
Figure 3: dsDNA Leakage in HeLa CT and ZMPSTE24 KO cells. Representative images of both cell lines used in this experiment. (A) HeLa control (CT) cell with green (Alexa Fluor 488 conjugated secondary) indicating dsDNA and red (Alexa Fluor 594) indicating the nucleus with Lamin B1. Scale bars: 100 &#181;m (40x magnification). (B) DNA leakage occurring in a ZMPSTE24 KO cell, with green (Alexa Fluor 488) indicating dsDNA and red (Alexa Fluor 594) indicating the nucleus. Scale bars: 100 &#181;m (40x magnification). (C) Larger microscope image of the HeLa control cells. (D) Larger microscope image of the HeLa ZMPSTE24 KO cells. In panels (C) and (D), the signals for nuclei and dsDNA are merged (40x magnification). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67719/67719fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For observing DNA leakage, an antibody against dsDNA is used in conjunction with a Lamin B1 antibody, which illuminates the nucleus of each cell (Figure 3A,B). A positive result for DNA leakage would be any green signal (dsDNA) outside of the red signal (Lamin B1) that is illuminating the nucleus. A negative result for DNA leakage is indicated when the green and red signals overlap each other, showing the dsDNA is retained within the nucleus. DNA leakage is not observed for the HeLa control cells (Figure 3A,C), whereas DNA leakage tends to occur in the vast majority of HeLa ZMPSTE24KO cells (Figure 3B,D). Interestingly, DNA leakage is seen in some ZMPSTE24 KO cells that display no visible nuclear blebbing.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67719/67719fig4.jpg" /> 
Figure 4: Detection of DNA and nuclear structure in GM637 cells. Representative images from immortalized cell line GM637, derived from an apparently healthy individual, to provide insight into the versatility of this IF protocol. (A) GM637 cell with blue (Alexa Fluor 405 conjugated secondary) indicating dsDNA and red (Alexa Fluor 594) indicating the nucleus with Lamin B1. Scale bars: 100 &#181;m (40x magnification). (B) Examples of nuclei displaying blebbing or no blebbing, with blebs indicated by white arrows. Scale bars: 100 &#181;m (40x magnification). Approximately 15% of these cells display blebbing, and no DNA leakage has been detected. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67719/67719fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
On a final note, we can demonstrate the versatility of this protocol by performing it on a second immortalized human cell line derived from an apparently healthy individual (GM637). For this cell line, a different secondary antibody was also used (Alexa Fluor 405 conjugated secondary) to detect the DNA (Figure 4).

Laminopathy disorders often lead to changes in the nuclear envelope, which may cause nuclear blebbing and leakage. This study presents an immunofluorescence method to visualize the nuclear lamina along with double-stranded DNA (dsDNA), providing a means to assess nuclear structure and integrity in mammalian cells.

Detection of Nuclear Blebbing and DNA Leakage in Mammalian Cells by Immunofluorescence

The nuclear lamina is a network of filaments underlying the nuclear membrane, composed of lamins and lamin-associated proteins. It plays critical roles in ...

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629 Views.  University of South Carolina. Laminopathy disorders often lead to changes in the nuclear envelope, which may cause nuclear blebbing and leakage. This study presents an immunofluorescence method to visualize the nuclear lamina along with double-stranded DNA (dsDNA), providing a means to assess nuclear structure and integrity in mammalian cells.

Video: Detection of Nuclear Blebbing and DNA Leakage in Mammalian Cells by Immunofluorescence

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 ...

Cancer Research

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Biochemistry

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

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 ...

Identifying DNA Damage in Prophase-Arrested Oocytes of Mice

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Developmental Biology

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Source: de la Pe&#241;a Avalos, B., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61447">Visualization of DNA Repair Proteins Interaction by Immunofluorescence.</a> J. Vis. Exp. (2020).
In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins &#947;H2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.

Oparta na immunofluorescencji wizualizacja interakcji białek naprawy DNA

Source: de la Pe&#241;a Avalos, B., et al., Visualization of DNA Repair Proteins Interaction by Immunofluorescence. J. Vis. Exp. (2020).
In this video, we ...

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Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks

Source: Popp, H. D., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/56617">Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks.</a> J. Vis. Exp. (2017)
In this video, we describe the immunofluorescence microscopy technique to detect specific DNA damage response proteins localized at the sites of DNA double-strand breaks in human mononuclear cells. The proteins are recognized by specific primary antibodies, which in turn bind to fluorophore-tagged secondary antibodies that fluoresce during microscopic visualization, enabling visualization of the proteins as distinct fluorescent foci within the cell nuclei.

Mikroskopia immunofluorescencyjna do analizy pęknięć dwuniciowych DNA

Source: Popp, H. D., et al. Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks. J. ...