We are grateful to the whole blood donors and all the health personnel who took the blood samples.

The study was approved by the ethical committee of Pisa University, and informed and signed consent was obtained from each donor.
1. Formation of &#947;H2AX and 53BP1 foci
<ol>
	<li>Preparation of samples and mutagenic treatment
	<ol>
		<li>Collect whole blood samples by venipuncture from healthy adult individuals in blood collection (e.g., Vacutainer) tubes containing lithium heparin as an anticoagulant.</li>
		<li>In order to guarantee proper blood sample preservation, start the procedure within 24 h of sampling.</li>
		<li>Add 300 &#181;L of the sample to a tube containing 4.7 mL of complete medium (0.5% penicillin-streptomycin, 0.75% phytohemagglutinin, 10% FBS previously inactivated, 88.75% RPMI 1640).</li>
		<li>Then add bleomycin sulfate to a final concentration of 5 &#181;g/mL. 
		CAUTION: Bleomycin sulfate is a mutagen. Avoid skin contact and inhalation. Prepare the solution and add the sample under a hood.</li>
		<li>For each sample, set up a negative control (absence of mutagen).</li>
		<li>Place the tube in a thermostat at 37 &#176;C for 2 h.</li>
	</ol>
	</li>
	<li>Fixation
	<ol>
		<li>Centrifuge samples at 540 x g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant and resuspend the pellet with vortex.</li>
		<li>Add 5 mL of hypotonic solution (2.87 g of KCl dissolved in 500 mL of deionized water) and 400 &#181;L of pre-fixative solution (5:3 acetic acid: methanol) to cause hemolysis of red blood cells.</li>
		<li>Centrifuge samples at 540 x g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant and resuspend the pellet in 5 mL of methanol at room temperature for at least 30 min to fix the cells.</li>
		<li>Alternatively, store the cells at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>Slides preparation
	<ol>
		<li>Centrifuge samples at 540 x g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant and resuspend the pellet in 5 mL of a 3:1 methanol: acetic acid solution. Repeat these steps one more time.</li>
		<li>At the end, centrifuge the solution again at 540 x g for 5 min at room temperature.</li>
		<li>Aspirate the supernatant again leaving enough solution (0.5 mL) to resuspend the pellet, pipette vigorously, drop the resuspended cell pellet on the slides, and air dry. Store the slides at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Immunofluorescence 
	NOTE: Immunofluorescence is an immunologic method for identifying specific cell targets using a primary antibody binding the target itself and a fluorescent secondary antibody binding the primary that allows localizing the target11. In this case, a mouse monoclonal anti-53BP1 (1:50) and a rabbit polyclonal anti-&#947;H2AX (1:50) are used as primary antibodies, while AlexaFluor568 anti-mouse (1:400) and DyLight488 anti-rabbit (1:200) are used as secondary antibodies, respectively.
	<ol>
		<li>Wash the slides two times in 50 mL of 1x PBS for 5 min in couplin jar (16 slides back-to-back).</li>
		<li>Then keep them for 30 min in blocking solution (10 mL of FBS, 10 mL of 10x PBS, 80 mL of deionized water, 300 &#181;L of Triton-X).</li>
		<li>Add to each slide 10 &#181;L of each of the two solutions containing the primary antibodies dissolved in the blocking solution. Cover the slides with paraffin tape and incubate at 4 &#176;C overnight.</li>
		<li>After incubation, perform three washing in 1x PBS for 5 min.</li>
		<li>Add to each slide 10 &#181;L of each of the two solutions containing the secondary antibodies dissolved in the blocking solution. Cover the slides with paraffin tape and incubate at room temperature for 2 h.</li>
		<li>Perform three washing in 1x PBS for 5 min.</li>
		<li>Add 2.5 &#181;L of antifade solution with DAPI on the coverslips before the assembly to counterstain the nuclei. 
		&#8203;NOTE: In order to assess foci kinetics, the procedure is the same as described from 1.1 to 1.4, noting that cell harvesting and the dual immunofluorescence are performed at 0 h, after 2 h post-bleomycin treatment and then, after having removed the mutagen, at 4 h, 6 h, and 24 h post-treatment.</li>
	</ol>
	</li>
</ol>
2. Analysis through a fluorescence microscope
NOTE: &#34;Fluorescence microscope&#34; refers to any microscope that uses fluorescence to generate an image. The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer wavelengths12. AlexaFluor568 and DyLight 488 absorb light of approximately 568 and 488 nm and emit light of 603 and 520 nm, respectively. Thus, they are visible as red or green fluorescence using a TRITC or a FITC filter, respectively.
<ol>
	<li>Score the presence of foci in each slide under a 100x immersion objective (Figure 1).</li>
	<li>Score 200 nuclei per slide and count the number of &#947;H2AX/53BP1 foci in each nucleus.</li>
	<li>Express results in terms of the average number of foci per total nuclei scored. These include both &#947;H2AX/53BP1 positive (showing at least one fluorescence signal) and negative (not showing any fluorescence signal) nuclei.</li>
</ol>

<ol>
	<li>Chatterjee, N., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+DNA+damage,+repair+and+mutagenesis.">Mechanisms of DNA damage, repair and mutagenesis.</a> Environmental and Molecular Mutagenesis. 58 (5), 235-263 (2017).</li><li>Aleksandrov, R., Hristova, R., Stoynov, S., Gospodinov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chromatin+response+to+double-strand+DNA+breaks+and+their+repair.">The chromatin response to double-strand DNA breaks and their repair.</a> Cells. 9 (8), 1853 (2020).</li><li>Jackson, S. P., Bartek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA-damage+response+in+human+biology+and+disease.">The DNA-damage response in human biology and disease.</a> Nature. 461 (7267), 1071-1078 (2009).</li><li>Dickey, J. S., 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=H2AX:+functional+roles+and+potential+applications.">H2AX: functional roles and potential applications.</a> Chromosoma. 118 (6), 683-692 (2009).</li><li>Her, J., Bunting, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+cells+ensure+correct+repair+of+DNA+double-strand+break.">How cells ensure correct repair of DNA double-strand break.</a> Journal of Biological Chemistry, Thematic Minireview. 293 (27), 10502-10511 (2018).</li><li>Kuo, L. K., Yang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#947;-H2AX+-+A+novel+biomarker+for+DNA+double-strand+breaks.">&#947;-H2AX - A novel biomarker for DNA double-strand breaks.</a> In Vivo. 22 (3), 305-310 (2008).</li><li>B&#225;rtov&#225;, E., Legartova, S., Dundr, M., Such&#225;nkov&#225;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+of+the+53BP1+protein+in+genome+protection:+structural+and+functional+characteristics+of+53BP1-dependent+DNA+repair.">A role of the 53BP1 protein in genome protection: structural and functional characteristics of 53BP1-dependent DNA repair.</a> Aging. 11 (8), 2488-2511 (2019).</li><li>Popp, H. D., Brendel, S., Hofman, W., Fabarius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+microscopy+of+&#947;H2AX+and+53BP1+for+analyzing+the+formation+and+repair+of+DNA+double-strand+breaks.">Immunofluorescence microscopy of &#947;H2AX and 53BP1 for analyzing the formation and repair of DNA double-strand breaks.</a> Journal of Visualized Experiments. 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F. P., Yong, 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=An+introduction+to+performing+immunofluorescence+staining.">An introduction to performing immunofluorescence staining.</a> Methods in Molecular Biology. 1897, 299-311 (2019).</li><li>Sanderson, M. J., Smith, I., Parker, I., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence microscopy. 10, (2014).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fixatives+for+immunostaining.">Cell fixatives for immunostaining.</a> Methods in Molecular Biology. , 55-61 (2010).</li><li>Jamur, M. C., Oliver, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeabilization+of+the+cell+membrane.">Permeabilization of the cell membrane.</a> Methods in Molecular Biology. 588, 63-66 (2010).</li><li>Hecht, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bleomycin:+New+perspectives+on+the+mechanism+of+action.">Bleomycin: New perspectives on the mechanism of action.</a> Journal of Natural Products. 63, 158-168 (2000).</li><li>Fei, P., El-Deiry, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P53+and+radiation+responses.">P53 and radiation responses.</a> Oncogene. 22, 5774-5783 (2003).</li><li>Mahaney, B. L., Meek, K., Lees-Miller, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+ionizing+radiation-induced+DNA+double-strand+breaks+by+non-homologous+end-joining.">Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining.</a> Biochemical Journal. 417 (3), 639-650 (2009).</li><li>Palla, V., 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=Gamma-H2AX:+Can+it+be+established+as+a+classical+cancer+prognostic+factor.">Gamma-H2AX: Can it be established as a classical cancer prognostic factor.</a> Tumor Biology. 39 (3), 1010428317695931 (2017).</li><li>Markov&#224;, E., Hillert, L., Malmgren, L., Persson, B. R. R., Belyaev, I. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwaves+from+GSM+mobile+telephones+affect+53BP1+and+gamma-H2AX+foci+in+human+lymphocytes+from+hypersensitive+and+healthy+persons.">Microwaves from GSM mobile telephones affect 53BP1 and gamma-H2AX foci in human lymphocytes from hypersensitive and healthy persons.</a> Environmental Health Perspectives. 113 (9), 1172-1177 (2005).</li><li>Scarpato, R., 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=Nuclear+damage+in+peripheral+lymphocytes+of+obese+and+overweight+Italian+children+as+evaluated+by+the+&#947;-H2AX+focus+assay+and+micronucleus+test.">Nuclear damage in peripheral lymphocytes of obese and overweight Italian children as evaluated by the &#947;-H2AX focus assay and micronucleus test.</a> The FASEB Journal. 25 (2), 685-693 (2018).</li><li>Shanbhag, N. 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=Early+neuronal+accumulation+of+DNA+double-strand+breaks+in+Alzheimer's+disease.">Early neuronal accumulation of DNA double-strand breaks in Alzheimer's disease.</a> Acta Neuropathologica Communication. 7 (1), 77 (2019).</li><li>Lassmann, 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=In+vivo+formation+of+gamma-H2AX+and+53BP1+DNA+repair+foci+in+blood+cells+after+radioiodine+therapy+of+differentiated+thyroid+cancer.">In vivo formation of gamma-H2AX and 53BP1 DNA repair foci in blood cells after radioiodine therapy of differentiated thyroid cancer.</a> Journal of Nuclear Medicine. 51 (8), 1318-1325 (2010).</li><li>Derlin, T., 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=Assessment+of+&#947;-H2AX+and+53BP1+foci+in+peripheral+blood+lymphocytes+to+predict+subclinical+hematotoxicity+and+response+in+somatostatin+receptor-targeted+radionuclide+therapy+for+advanced+gastroenteropancreatic+neuroendocrine+tumors.">Assessment of &#947;-H2AX and 53BP1 foci in peripheral blood lymphocytes to predict subclinical hematotoxicity and response in somatostatin receptor-targeted radionuclide therapy for advanced gastroenteropancreatic neuroendocrine tumors.</a> Cancers (Basel). 13 (7), 1516 (2021).</li><li>Djuzenova, C. S., 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=Radiosensitivity+in+breast+cancer+assessed+by+the+histone+&#947;-H2AX+and+53BP1+foci.">Radiosensitivity in breast cancer assessed by the histone &#947;-H2AX and 53BP1 foci.</a> Radiation Oncology. 24, 8-98 (2013).</li><li>Atkinson, J., Bezak, E., Kempson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+DNA+double-strand+breaks+-+are+we+there+yet.">Imaging DNA double-strand breaks - are we there yet.</a> Nature Reviews in Molecular Cell Biology. 23, 579-580 (2022).</li></ol>

The authors have nothing to disclose.

Immunofluorescence analysis of &#947;H2AX and 53BP1 foci is a suitable method for assessing genomic damage in interphase nuclei of a cell system. This procedure has several critical points that can affect the outcome of the experiments, mainly, the agents used in fixation and permeabilization, the type of antibodies and their dilution factors, and the concentration of the mutagen.
The maintenance of protein integrity is fundamental since the immunofluorescence method expects to identify antige...

DNA damage is continuously induced by agents that can be endogenous, such as ROS generated by cellular oxidative metabolism, or exogenous, both chemicals and physical1. Among the most harmful lesions, double-strand breaks (DSBs) play a fundamental role in contributing to genomic instability, since they cause chromosome aberrations that in turn can initiate the carcinogenesis process. Thus, cells are provided with complex and efficient mechanisms of DSBs repairing2.
When a DSB occurs, the cell triggers the DNA damage response (DDR) where, together with the MRE11/RAD50/NBS1 complex, ATM or ATR kinases are recruited to activate other proteins that slow down or stop the cell cycle3. An essential target of these kinases is histone H2AX, which is phosphorylated on Ser-139 within a few megabases from the DSBs (namely &#947;H2AX), thereby allowing the recruitment of several repair factors such as, among others, BRCA1 and p53 binding protein 1 (53BP1)3. Later, one pathway among homologous recombination (HR), non-homologous end joining (NHEJ), or single-strand annealing (SSA) is triggered to repair the DSBs4,5. Therefore, 53BP1 is involved in dictating the choice between HR or NHEJ, mainly promoting the activation of NHEJ rather than HR6. Moreover, both the phosphorylated form of H2AX histone and 53BP1 can form foci at the sites of DSBs. As these foci persist until the integrity of the double strand is restored, assessing the appearance/disappearance of &#947;H2AX or 53BP1 foci within a time interval is considered a useful method to evaluate the occurrence and repair of DSBs in a cell system6,7. However, according to the molecular processes above described, since &#947;H2AX and 53BP1 foci are expected to co-localize near the DSBs during DDR8,9, it can be useful to detect concurrently the presence of these markers in a dual immunofluorescence.
Thus, the aim of this manuscript was to evaluate the suitability of the simultaneous quantification of &#947;H2AX and 53BP1 foci to assess the genomic damage induced in human peripheral lymphocytes by the radiomimetic agent bleomycin. Using the same methodology, we also attempted to delineate repair kinetics of bleomycin-induced DSBs according to a previously set up experimental procedure10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AlexaFluor 568 goat anti-mouse IgG (&#947;1)</td>
			<td>Invitrogen</td>
			<td>A21124</td>
			<td>53BP1 secondary antibody</td>
		</tr>
		<tr>
			<td>Bleoprim</td>
			<td>Sanofi</td>
			<td></td>
			<td>bleomycin sulfate (mutagen)</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin solution 100X</td>
			<td>Euroclone</td>
			<td>ECB3001D</td>
			<td>antibiotics for culture medium</td>
		</tr>
		<tr>
			<td>PBS 10X</td>
			<td>Termofisher</td>
			<td>14200075</td>
			<td>Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Euroclone</td>
			<td>EC20180L</td>
			<td>Fetal Bovine Serum for immunofluorescence</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) DyLight 488 Coniugated</td>
			<td>Termofisher</td>
			<td>#35552</td>
			<td>&#947;H2AX secondary antibody</td>
		</tr>
		<tr>
			<td>Mouse anti-53BP1 monoclonal antibody</td>
			<td>Merck</td>
			<td>MAB 3802</td>
			<td>53BP1 primary antibody</td>
		</tr>
		<tr>
			<td>Labophot 2</td>
			<td>Nikon</td>
			<td></td>
			<td>Fluorescence microscope</td>
		</tr>
		<tr>
			<td>P-histone H2AX (Ser139) rabbit antibody</td>
			<td>Cell Signaling</td>
			<td>#2577</td>
			<td>&#947;H2AX primary antibody</td>
		</tr>
		<tr>
			<td>Phytohemoagglutinin</td>
			<td>Termofisher</td>
			<td>R30852801</td>
			<td>component of culture medium</td>
		</tr>
		<tr>
			<td>Prolong gold antifade reagent with DAPI</td>
			<td>Cell Signaling</td>
			<td>#8961</td>
			<td>Antifade solution with DAPI for counterstaining</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Euroclone</td>
			<td>ECB9006L</td>
			<td>Culture medium</td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td>Nonionic detergent for permeabilization</td>
		</tr>
	</tbody>
</table>

dual immunofluorescence of &#947;h2ax and 53bp1 in human peripheral lymphocytes

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, including cancer. The cell is, therefore, provided with complex mechanisms to repair DSBs, and these pathways involve histone H2AX in its phosphorylated form at Ser-139 (namely &#947;H2AX) and p53 binding protein 1 (53BP1). As both proteins can form foci at the sites of DSBs, identification of these markers is considered a suitable method to study both DSBs and their kinetics of repair. According to the molecular processes that lead to the formation of &#947;H2AX and 53BP1 foci, it could be more useful to investigate their co-localization near the DSBs in order to set up an alternative approach that allows quantifying DSBs by the simultaneous detection of two DNA damage markers. Thus, this protocol aims to assess the genomic damage induced in human lymphocytes by the radiomimetic agent bleomycin through the presence of &#947;H2AX and 53BP1 foci in a dual immunofluorescence. Using this methodology, we also delineated the variation in the number of &#947;H2AX and 53BP1 foci over time, as a preliminary attempt to study the repair kinetics of bleomycin-induced DSBs.

Data obtained by the fluorescence microscope analysis of peripheral lymphocytes allow us to evaluate three main aspects: the effectiveness of bleomycin treatment in increasing the number of &#947;H2AX and 53BP1 foci (and thus of DSBs) due to its mutagenic effect, at what extent both foci co-localized at the site of DSBs, and the time-course of &#947;H2AX and 53BP1 foci to delineate the repair kinetics of bleomycin-induced DSBs. As expected, a very higher frequency of both &#947;H2AX and 53BP1 foci was observed between un...

Watch this Scientific Journal Video about Dual Immunofluorescence of ÃŽÂ³H2AX and 53BP1 in Human Peripheral Lymphocytes at JoVE.com

Dual Immunofluorescence of &#947;H2AX and 53BP1 in Human Peripheral Lymphocytes

This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of &#947;H2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

Double strand breaks (DSBs) are one of the most severe lesions that can occur in cell nuclei, and, if not repaired, they can lead to severe outcomes, ...

dual-immunofluorescence-h2ax-53bp1-human-peripheral

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1.3K Views.  University of Pisa. This protocol presents a method to assess the formation and repair of DNA double-strand breaks through the simultaneous detection of γH2AX and 53BP1 foci in interphase nuclei of bleomycin-treated human peripheral lymphocytes.

Video: Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ...

ATAC-seq Assay with Low Mitochondrial DNA Contamination from Primary Human CD4+ T Lymphocytes

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible chromatin. In this paper we present an improved ATAC-seq protocol that reduces contaminating mitochondrial DNA reads. While previous ATAC-seq protocols have struggled with an average of 50% contaminating mitochondrial DNA reads, the optimized&#160;lysis buffer introduced in this protocol reduces mitochondrial DNA contamination to an average of 3%. This improved ATAC-seq protocol allows for a near 50% reduction in the sequencing cost. We demonstrate how these high-quality ATAC-seq libraries can be prepared from activated CD4+ lymphocytes, providing step-by-step instructions from CD4+ lymphocyte isolation from whole blood through data analysis. This improved ATAC-seq protocol has been validated in a wide range of cell types and will be of immediate use to researchers studying chromatin accessibility.

Here, we present a protocol to perform an assay for transposase-accessible chromatin sequencing (ATAC-seq) on activated CD4+ human lymphocytes. The protocol has been modified to minimize contaminating mitochondrial DNA reads from 50% to 3% through the introduction of a new lysis buffer.

ATAC-seq has become a widely used methodology in the study of epigenetics due to its rapid and simple approach to mapping genome-wide accessible ...

Navigating MARRVEL, a Web-Based Tool that Integrates Human Genomics and Model Organism Genetics Information

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is pathogenic, one must query many databases to determine whether the gene of interest is linked to a genetic disease, whether the specific variant has been reported before, and what functional data is available in model organism databases that may provide clues about the gene&#8217;s function in human. MARRVEL (Model organism Aggregated Resources for Rare Variant ExpLoration) is a one-stop data collection tool for human genes and variants and their orthologous genes in seven model organisms including in mouse, rat, zebrafish, fruit fly, nematode worm, fission yeast, and budding yeast. In this Protocol, we provide an overview of what MARRVEL can be used for and discuss how different datasets can be used to assess whether a variant of unknown significance (VUS) in a known disease-causing gene or a variant in a gene of uncertain significance (GUS) may be pathogenic. This protocol will guide a user through searching multiple human databases simultaneously starting with a human gene with or without a variant of interest. We also discuss how to utilize data from OMIM, ExAC/gnomAD, ClinVar, Geno2MP, DGV and DECHIPHER. Moreover, we illustrate how to interpret a list of ortholog candidate genes, expression patterns, and GO terms in model organisms associated with each human gene. Furthermore, we discuss the value protein structural domain annotations provided and explain how to use the multiple species protein alignment feature to assess whether a variant of interest affects an evolutionarily conserved domain or amino acid. Finally, we will discuss three different use-cases of this website. MARRVEL is an easily accessible open access website designed for both clinical and basic researchers and serves as a starting point to design experiments for functional studies.

Here, we present a protocol to access and analyze many human and model organism databases efficiently. This protocol demonstrates the use of MARRVEL to analyze candidate disease-causing variants identified from next-generation sequencing efforts.

Through whole-exome/genome sequencing, human geneticists identify rare variants that segregate with disease phenotypes. To assess if a specific variant is ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

Characterizing Mutational Load and Clonal Composition of Human Blood

Hematopoietic stem and progenitor cells (HSPCs) gradually accumulate DNA mutations during a lifespan, which can contribute to age-associated diseases such as leukemia. Characterizing mutation accumulation can improve understanding of the etiology of age-associated diseases. Presented here is a method to catalogue somatic mutations in individual HSPCs, which is based on whole-genome sequencing (WGS) of clonal primary cell cultures. Mutations that are present in the original cell are shared by all cells in the clonal culture, whereas mutations acquired in vitro after cell sorting are present in a subset of cells. Therefore, this method allows for accurate detection of somatic mutations present in the genomes of individual HSPCs, which accumulate during life. These catalogues of somatic mutations can provide valuable insights into mutational processes active in the hematopoietic tissue and how these processes contribute to leukemogenesis. In addition, by assessing somatic mutations that are shared between multiple HSPCs of the same individual, clonal lineage relationships and population dynamics of blood populations can be determined. As this approach relies on in vitro expansion of single cells, the method is limited to hematopoietic cells with sufficient replicative potential.

Somatic mutation patterns in cells reflect previous mutagenic exposure and can reveal developmental lineage relationships. Presented here is a methodology to catalogue and analyze somatic mutations in individual hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) gradually accumulate DNA mutations during a lifespan, which can contribute to age-associated diseases such ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Microsatellite DNA Genotyping and Flow Cytometry Ploidy Analyses of Formalin-fixed Paraffin-embedded Hydatidiform Molar Tissues

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are two types of HM based on microscopic morphological evaluation, complete HM (CHM) and partial HM (PHM). These can be further subdivided based on the parental contribution to the molar genomes. Such characterization of HM, by morphology and genotype analyses, is crucial for patient management and for the fundamental understanding of this intriguing pathology. It is well documented that morphological analysis of HM is subject to wide interobserver variability and is not sufficient on its own to accurately classify HM into CHM and PHM and distinguish them from hydropic non-molar abortions. Genotyping analysis is mostly performed on DNA and tissues from formalin-fixed paraffin-embedded (FFPE) products of conception, which have less than optimal quality and may consequently lead to wrong conclusions. In this article, detailed protocols for multiplex genotyping and flow cytometry analyses of FFPE molar tissues are provided, along with the interpretation of the results of these methods, their troubleshooting, and integration with the morphological evaluation, p57KIP2 immunohistochemistry, and fluorescence in situ hybridization (FISH) to reach a correct and robust diagnosis. Here, the authors share the methods and lessons learned in the past 10 years from the analysis of approximately 400 products of conception.

Hydatidiform moles are abnormal human pregnancies with heterogeneous aetiologies that can be classified according to their morphological features and parental contribution to the molar genomes. Here, protocols of multiplex microsatellite DNA genotyping and flow cytometry of formalin-fixed paraffin-embedded molar tissues are described in detail, together with results&#8217; interpretation and integration.

Hydatidiform mole (HM) is an abnormal human pregnancy characterized by excessive trophoblastic proliferation and abnormal embryonic development. There are ...