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 t...

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/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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.&#160;</td>
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		<tr>
			<td>36.5% Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td>Fixation Reagent.&#160;</td>
		</tr>
		<tr>
			<td>75 cm 2 Flasks</td>
			<td>Corning</td>
			<td>430725U</td>
			<td>For cell culture.&#160;</td>
		</tr>
		<tr>
			<td>Bovine Serum Albinum (BSA)</td>
			<td>Fisher Scientific</td>
			<td>9048-46-8</td>
			<td>Antibody dilution buffer.&#160;</td>
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			<td>DMEM with 1 g/L glucose, L-glutamine &#38; sodium pyruvate</td>
			<td>Corning</td>
			<td>10-014-CV</td>
			<td>For cell culture.&#160;</td>
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			<td>Ethanol (200 Proof)</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td>For dehydration of samples before mounting, diluted to make multiple concentrations.&#160;</td>
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		<tr>
			<td>EVOS FL Digital Inverted Fluorescence Microscope</td>
			<td>Fisher Scientific</td>
			<td>12-563-460</td>
			<td>Imaging.&#160;</td>
		</tr>
		<tr>
			<td>Fish Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>G7041</td>
			<td>Antibody dilution buffer.&#160;</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Mounting medium to prevent photobleaching.&#160;</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.&#160;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15-140-122</td>
			<td>For cell culture.&#160;</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.&#160;</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.&#160;</td>
		</tr>
		<tr>
			<td>Tissue Culture Treated 6-well Flat Bottom Plates</td>
			<td>Falcon</td>
			<td>353046</td>
			<td>For cell culture.&#160;</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.&#160;</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>9005-64-5</td>
			<td>Cell Permeation reagent.&#160;</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<...

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 ...

detection-nuclear-blebbing-dna-leakage-mammalian-cells

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596 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

Trypsinizing and Subculturing Mammalian Cells

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in cell death. The first step in subculturing is to detach cells from the surface of the primary culture vessel by trypsinization or mechanical means. The resultant cell suspension is then subdivided, or reseeded, into fresh cultures. Secondary cultures are checked for growth and fed periodically, and may be subsequently subcultured to produce tertiary cultures. The time between passaging of cells varies with the cell line and depends on the growth rate.

As cells reach confluency, they must be subcultured or passaged. This video will demonstrate a procedure for subculturing both adherent and suspension cells.

As cells reach confluency, they must be subcultured or passaged. Failure to subculture confluent cells results in reduced mitotic index and eventually in ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to immortalized cell lines. However, the post-mitotic nature of primary neurons prevents effective heterologous protein expression using common procedures such as electroporation or chemically-mediated transfection. Thus, other techniques must be employed in order to effectively express proteins in these non-dividing cells.
In this article, we describe the steps required to perform intranuclear injections of cDNA constructs into dissociated adult sympathetic neurons. This technique, which has been applied to different types of neurons, can successfully induce heterologous protein expression. The equipment essential for the microinjection procedure includes an inverted microscope to visualize cells, a glass injection pipet filled with cDNA solution that is connected to a N2(g) pressure delivery system, and a micromanipulator. The micromanipulator coordinates the injection motion of microinjection pipet with a brief pulse of pressurized N2 to eject cDNA solution from the pipet tip. 
This technique does not have the toxicity associated with many other transfection methods and enables multiple DNA constructs to be expressed at a consistent ratio. The low number of injected cells makes the microinjection procedure well suited for single cell studies such as electrophysiological recordings and optical imaging, but may not be ideal for biochemical assays that require a larger number of cells and higher transfection efficiencies. Although intranuclear microinjections require an investment of equipment and time, the ability to achieve high levels of heterologous protein expression in a physiologically relevant environment makes this technique a very useful tool to investigate protein function.

Direct intranuclear injection of cDNA is an effective transfection technique for post-mitotic cells. This method provides high levels of heterologous protein expression from single or multiple cDNA constructs and enables protein function to be studied in a physiologically relevant environment with a variety of single cell assays.

Primary neuronal cell cultures are valuable tools to study protein function since they represent a more biologically relevant system compared to ...

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 ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

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

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

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Trypsinizing and Subculturing Mammalian Cells

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

Intranuclear Microinjection of DNA into Dissociated Adult Mammalian Neurons

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

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

Analysis of Cell Cycle Position in Mammalian Cells

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation