We thank the Dungrawala lab members for their input and feedback. This work was funded by NIH grant R35GM137800 to HD.

This protocol (Figure 1) primarily utilizes adherent U2OS cancer cell lines but is adaptable to a wide variety of adherent and suspension cells grown in tissue culture. The solutions and buffers used in this study are detailed in Table 1. The reagents and equipment used are listed in the Table of Materials.
1. Preparation of materials
<ol>
	<li>Prior to the day of the experiment, place the provided bottle with low melting agarose (1% low melting agarose in PBS) in a hot water bath for 5-10 min to ensure the agarose has completely melted. 
	NOTE: Do not heat the bottle in the microwave.</li>
	<li>Once melted, quickly aliquot 100 &#181;L of the agarose to 1.5 mL centrifuge tubes as required and store the tubes at 4 &#176;C. The solidified agarose can be stored at 4 &#176;C for up to 1 year.</li>
	<li>On the day of the experiment, remove the required number of centrifuge tubes from the 4 &#176;C storage. Melt the agarose by placing the tubes in a heat block or water bath at 42 &#176;C. 
	NOTE: Place the tubes at 42 &#176;C prior to harvesting the cells to ensure the agarose is completely melted for resuspension (step 3.7).</li>
	<li>Prepare fresh lysis buffer and TAE buffer on the day of the experiment and store them at room temperature and 4 &#176;C, respectively, until ready to use. In addition, aliquot 1x PBS in 15 mL conical tube and place it in the fridge to use in the resuspension step (step 3.5).</li>
	<li>Mark the sample identifications on the frosted end of the 2-well comet slides and store them at room temperature until ready to use.</li>
</ol>
2. Preparation of samples
<ol>
	<li>Prior to the day of the experiment, seed the cells in a 6-well dish and culture overnight at 37 &#176;C with 5% CO2. For U2OS cells, approximately 2-3 x 105 cells were seeded for each sample. 
	NOTE: Cell numbers might need to be adjusted depending on the cell type used for analyses.</li>
	<li>On the day of the experiment, treat cells with the conditions as per the experimental design. As a positive control to demonstrate replication-dependent break induction, treat cells with 1 &#181;M CPT for 1 h. CPT is a topoisomerase-1 inhibitor that induces double-strand breaks in a replication-dependent manner14.</li>
</ol>
3. Cell resuspension and lysis
<ol>
	<li>Aspirate cell culture media and rinse twice with 1x PBS.</li>
	<li>Add 300 &#181;L of trypsin to each well. Incubate the dish in a tissue culture incubator for 2-3 min. 
	NOTE: Prolonged incubation in trypsin can generate spurious results during comet analyses.</li>
	<li>Add 700 &#181;L of cell culture media to each well. Resuspend gently and transfer the samples to 1.5 mL centrifuge tubes. 
	NOTE: Counting cells at this step is recommended to obtain the appropriate cell density in step 3.6 to reduce the frequency of overlapping tails during analysis.</li>
	<li>Centrifuge the cell suspension at 2400 x g&#160;for 5 min at room temperature.</li>
	<li>Aspirate the media and resuspend the cells in 1 mL of cold 1x PBS.</li>
	<li>Repeat step 3.4 and step 3.5 twice and resuspend the cells in cold 1x PBS at a cell density of 2 x 105 cells per ml. 
	NOTE: Assessing cell viability at this step is recommended to ensure an adequate number of viable cells are present. At least 90% viability is recommended.</li>
	<li>Resuspend 10 &#181;L of the cell suspension in 100 &#181;L aliquot of melted agarose, and using the same pipette tip, mix gently and spread the mixture evenly into one well of the slide. Repeat this step for all samples as needed. 
	NOTE: Avoid removing all aliquots from 42 &#176;C water bath at once to prevent the agarose from solidifying. This step should preferably be performed by the water bath, and each sample is resuspended and spread in a staggered manner. Avoid introducing bubbles while spreading the agarose on the slide since the air pockets will affect the migration of the comet tails during electrophoresis (step 4.5) and introduce cracks in the drying step (step 5.3)</li>
	<li>Store the slides at 4 &#176;C in the dark for 15-20 min to ensure the agarose has completely solidified (Figure 2A).</li>
	<li>Place the slides in Coplin jars and immerse the slides in lysis buffer for 1 h at room temperature.</li>
	<li>Rinse the slides once with cold TAE buffer and immerse the slides in cold TAE buffer for 30 min at 4 &#176;C.</li>
</ol>
4. Electrophoresis
<ol>
	<li>Setup the electrophoretic chamber on a flat surface and level the system using the four leveling knobs on the edges of the apparatus. Insert cold packs in the bottom chamber to keep the solution cold during electrophoresis.</li>
	<li>Pour approximately ~850 mL of cold 1x TAE buffer into the apparatus.</li>
	<li>Remove the slides from 4 &#176;C and place them on the electrophoresis tray in the proper orientation to allow for the migration of broken DNA. 
	NOTE: The tray holds ten 2-well slides. Place the slides in the center of the tray if working with less number of samples. If working with an odd number of slides, include a blank slide such that each well accommodates 2 slides.</li>
	<li>Gently place the provided slide tray overlay on top of the tray to cover the slides. 
	NOTE: For proper operation of the apparatus, the volume of the TAE buffer should not be lower than the bottom surface of the overlay and not exceed the top surface of the overlay.</li>
	<li>After connecting the cables, set the power at 21 V and perform electrophoresis (1 V/cm) for 40 min. 
	NOTE: The electrophoresis time may need to be adjusted depending on the cell type used for analyses. Long tails in untreated samples indicate excessive runtime. Conversely, short tails in positive control samples may reflect insufficient runtime.</li>
</ol>
5. Staining
<ol>
	<li>After completion of electrophoresis, remove the slides from the apparatus and immerse them in DNA precipitation solution for 30 min at room temperature.</li>
	<li>Discard the DNA precipitation solution and immerse the slides in 70% ethanol solution for 30 minu at room temperature.</li>
	<li>Discard the 70% ethanol solution. Remove the slides and dry them in a hybridization oven at 45 &#176;C for 30 min in the dark (Figure 2B). 
	NOTE: Slides can be left in a dark, dry place overnight for the drying step.</li>
	<li>Incubate the wells with 100 &#181;L of 1x SYBR Green solution for 30 min at room temperature in the dark.</li>
	<li>Decant the SYBR Green solution by tilting the slides and removing the excess by gently dabbing the edge of the well with a lint-free wipe.</li>
	<li>Place the slides in a drawer and allow to dry for 30 min.</li>
	<li>Proceed with imaging of the slides.</li>
</ol>
6. Imaging and analyses
<ol>
	<li>Setup the microscope for imaging purposes. 
	NOTE: All images were acquired using a epifluorescence microscope. 10x magnification with 0.45 numerical aperture was used with an exposure time of 1/40s and a gain of +6dB. The capturing mode of monochrome 8bit was utilized with 2 x 2 binning and black balance correction.</li>
	<li>Place the slides on the microscope stage and begin imaging using the FITC filter. 
	NOTE: Use the untreated sample first to test exposure time. Most cells do not exhibit comet tails since DNA is more compact. Switch to the well with CPT-treated cells and image using the same exposure time. Comet tails with increased lengths will be observed in this sample due to the migration of broken DNA ends.</li>
	<li>Acquire images for at least 100 comets per sample using the same exposure time across three technical replicates. 
	NOTE: Comet tails at the end of coverslips tend to migrate unevenly and act as outliers in the sample distribution. Most of the comets captured for analyses are present close to the center of the coverslip (Figure 3).</li>
	<li>Export images and score the comets either manually (using Image J) or by using freely available comet scoring software. Comets that are individually distinguishable and fully contained in the field of view are selected for scoring. Overlapping or partially visible comets are not considered for further analyses (Figure 4). 
	NOTE: Users can score comets on Image J using the following plugin: https://cometbio.org/index.html. Details for scoring comets using CometScore software are included in&#160;Figure 5.</li>
	<li>Plot tail moments acquired from the analyses as box and whisker plots depicting 10-90 percentile values. Perform normality tests to determine whether parametric, or non-parametric tests should be utilized for statistical analyses. 
	NOTE: In most cases, non-parametric tests are applied using the Mann-Whitney test if comparing two samples only or the Kruskal-Wallis test with Dunn&#39;s multiple comparisons if comparing three or more samples.</li>
</ol>

Investigating the Extent of DNA Breaks Occurring During DNA Replication 

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay:+A+method+to+measure+DNA+damage+in+individual+cells.">The comet assay: A method to measure DNA damage in individual cells.</a> Nat Protoc. 1 (1), 23-29 (2006).</li><li>De Lapuente, J., 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+comet+assay+and+its+applications+in+the+field+of+ecotoxicology:+A+mature+tool+that+continues+to+expand+its+perspectives.">The comet assay and its applications in the field of ecotoxicology: A mature tool that continues to expand its perspectives.</a> Front Genet. 6, 180 (2015).</li><li>Azqueta, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+comet+assay+in+human+biomonitoring:+An+hcomet+perspective.">Application of the comet assay in human biomonitoring: An hcomet perspective.</a> Mutat Res Rev Mutat Res. 783, 108288 (2020).</li><li>Speit, G., Hartmann, 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+comet+assay:+A+sensitive+genotoxicity+test+for+the+detection+of+DNA+damage.">The comet assay: A sensitive genotoxicity test for the detection of DNA damage.</a> Methods Mol Biol. 291, 85-95 (2005).</li><li>Collins, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+DNA+modifications+with+the+comet+assay:+A+compendium+of+protocols.">Measuring DNA modifications with the comet assay: A compendium of protocols.</a> Nat Protoc. 18 (3), 929-989 (2023).</li><li>Rapp, A., Hausmann, M., Greulich, K. 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The authors declare no competing interests.

This protocol outlines a NC assay for assessing DNA breaks in human cells undergoing active replication. The protocol is relatively simple to perform, and researchers can readily adapt it to high pH conditions for alkaline analyses8. It includes two critical steps, as described in steps 3.7 and 4.5. In step 3.7, it is essential to spread the melted agarose with cells uniformly across the well to prevent comets from overlapping during analysis. Ensure that cellular aggregates are dissolved before r...

The comet assay is a gel electrophoresis method used to detect DNA breaks at the single-cell level. It relies on the principle that open-ended DNA breaks migrate under electrophoretic conditions, while intact DNA remains largely static. Broken DNA migrates because breaks result in the relaxation of supercoiled DNA, causing its gradual spatial relocation towards the anode in the electrophoretic chamber, which results in a comet-like appearance observed by immunofluorescence1,2. The extent of DNA damage is then measured by quantifying the amount of broken DNA that has migrated relative to the compact DNA.
The two most commonly used comet assay methods are the alkaline comet (AC) assay and the neutral comet (NC) assay. The AC assay is performed under denaturing conditions using a high alkaline pH solution, while the NC assay is carried out in a solution with neutral pH. Both NC and AC assays can reliably detect DNA breaks that occur in the nucleus. However, the alkaline version is also advantageous for detecting alkali-labile sites3. Both formats of the assay require the use of lysis conditions to ensure that DNA is free of proteins prior to electrophoresis.
This assay is a simple method for detecting DNA breaks and offers several unique advantages for determining cellular DNA damage. The method is relatively easy to set up and requires reagents that can either be prepared in the laboratory or purchased from commercial vendors. As a single-cell resolution technique, the assay requires very little starting material. Notably, the NC assay can measure breaks with high sensitivity, reported to detect between 50 and 10,000 breaks per cell4. The versatility of this technique is demonstrated by its wide range of applications, including ecotoxicology5, human biomonitoring6, and genotoxicity studies7. The assay can also be reliably used across various cell types and adapted for high-throughput assays8 and for assessing breaks at specific genomic regions9. Thus, this assay serves as a rapid and reliable technique for investigating break formation at the single-cell level.
The process of DNA replication is constantly challenged by several endogenous and exogenous stressors10,11. Stalling of replisomes due to such lesions can cause DNA breaks and result in heightened genome instability. DNA breaks also often arise as replication intermediates to facilitate DNA repair12. Additionally, several chemotherapeutic agents induce replication-dependent double-strand breaks (DSBs), such as camptothecin (CPT)13,14,15and PARP inhibitors16,17. Thus, this assay serves as a powerful methodology to study the nature of DNA lesions, assess the processing of replication forks, and investigate the therapeutic potential of DNA-damaging agents. Adaptations of the assay that involve labeling newly synthesized DNA with BrdU have been useful for studying replication-associated stress phenotypes18,19,20,21. Notably, the NC assay is a reliable method for studying DNA break induction in the context of replication, as demonstrated in studies involving the characterization of fork proteins22,23, analysis of replication intermediates24,25, and investigation of transcription-replication conflicts in genome maintenance26,27. Herein, we outline a NC assay protocol with the goal of quantifying DNA breaks during replication in human cells. This protocol can be readily implemented by researchers interested in assessing replication stress-associated damage in a wide variety of dividing human cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1x DPBS</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Camptothecin</td>
			<td>Selleckchem</td>
			<td>S1288</td>
			<td></td>
		</tr>
		<tr>
			<td>Comet LMAgarose (LMA)</td>
			<td>R&#38;D systems</td>
			<td>4250-050-02</td>
			<td></td>
		</tr>
		<tr>
			<td>CometAssay Electrophoresis System II</td>
			<td>R&#38;D systems</td>
			<td>4250-050-ES</td>
			<td>Includes electrophoresis tank, safety lid, cables, 2/20 wells slide trays and slide tray overlay</td>
		</tr>
		<tr>
			<td>CometScore software</td>
			<td>TriTek</td>
			<td></td>
			<td>open-sourced</td>
		</tr>
		<tr>
			<td>CometSlide</td>
			<td>R&#38;D systems</td>
			<td>4250-050-03</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO&#160;</td>
			<td>FisherSci</td>
			<td>D128-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Keyence</td>
			<td>BZ-X810</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum - Premium</td>
			<td>Bio-Techne</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 10.0</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>hTERT-RPE-1 cells</td>
			<td>ATCC</td>
			<td>CRL-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydroxyurea</td>
			<td>Millipore Sigma</td>
			<td>H8627</td>
			<td></td>
		</tr>
		<tr>
			<td>PARP inhibitor Olaparib</td>
			<td>Selleckchem</td>
			<td>S8096</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac&#160;</td>
			<td>FisherSci</td>
			<td>FB300Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Surface Treated Sterile Tissue Culture Plate</td>
			<td>FisherSci</td>
			<td>FB012927</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green I Nucleic Acid Gel Stain (10,000x)</td>
			<td>FisherSci</td>
			<td>S7567</td>
			<td></td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
		<tr>
			<td>UVP&#160;HB-1000 Hybridization Incubator</td>
			<td>FisherSci</td>
			<td>UVP95003001</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of dna breaks in dividing human cells by neutral comet assay

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during genome duplication can stall replisomes and convert replication forks into double-strand breaks. If left unrepaired, these toxic DNA breaks can trigger chromosomal rearrangements, leading to heightened genome instability and an increased likelihood of cellular transformation. Additionally, cancer cells exhibit persistent replication stress, making the targeting of replication fork vulnerabilities in tumor cells an attractive strategy for chemotherapy. A highly versatile and powerful technique to study DNA breaks during replication is the comet assay. This gel electrophoresis technique reliably detects the induction and repair of DNA breaks at the single-cell level. Herein, a protocol is outlined that allows investigators to measure the extent of DNA damage in mitotically dividing human cells using fork-stalling agents across multiple cell types. Coupling this with automated comet scoring facilitates rapid analysis and enhances the reliability in studying induction of DNA breaks.

Analysis of break induction by replication stress reagents 
U2OS cells were treated with DMSO, 4 of mM hydroxyurea (HU), or 100 nM of CPT for 4 h and analyzed for break accumulation by the NC assay (Figure 6A). While CPT induces breaks during S-phase by blocking topoisomerase-113, HU-induced depletion of nucleotide pools stalls replication forks that are progressively converted into DSBs28. The data indicates that exposure...

Author Spotlight: Unveiling the Role of SNF2L in Replication Fork Stability and Genome Duplication

This protocol is designed to investigate the extent of DNA damage occurring during DNA replication. Under neutral conditions, the induction of DNA breaks can be readily assessed within a short time frame. Additionally, the protocol is adaptable to other cell types and various replication stress reagents.

Detection of DNA Breaks in Dividing Human Cells by Neutral Comet Assay

DNA replication is constantly challenged by a wide variety of endogenous and exogenous stressors that can damage DNA. Such lesions encountered during ...

detection-of-dna-breaks-in-dividing-human-cells-by-neutral-comet-assay

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

370 Views.  University of South Florida, Tampa. This protocol is designed to investigate the extent of DNA damage occurring during DNA replication. Under neutral conditions, the induction of DNA breaks can be readily assessed within a short time frame. Additionally, the protocol is adaptable to other cell types and various replication stress reagents.

Video: Author Spotlight: Unveiling the Role of SNF2L in Replication Fork Stability and Genome Duplication

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Detection of DNA Breaks in Dividing Human Cells by Neutral Comet Assay

Summary

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Chapters in this video

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining