The work reported in this publication was, in part, supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under award number: 1R41ES030274. The content is solely the authors&#39; responsibility and does not necessarily represent the official view of the National Institutes of Health.

Commercially available blood samples were used in the present study. At our institution, Institutional Review Board approval is not needed for the use of commercially available blood.
1. Preparation of materials for the comet assay
<ol>
	<li>Preparation of the microscope slides
	<ol>
		<li>Pour 1% (w/v) normal melting point agarose [dissolved in double-distilled water (ddH2O)] in a 50 mL tube and microwave to dissolve the agarose in the ddH2O. Store at 37 &#176;C to prevent solidification prior to coating slides. Should solidification occur, discard and prepare fresh.</li>
		<li>Pre-coat microscope slides by dipping the slides into the 50 mL tube containing 1% (w/v) normal melting point agarose.</li>
		<li>Wipe the back of the slides quickly after dipping the slides. 
		NOTE: Failure to wipe the back of the slides properly will increase the background noise of the slides during the analysis step by microscope.</li>
		<li>Label the coated slide with a permanent marker on the bottom-right corner of the frosted section (Figure 3A). This shows which side of the slide is pre-coated.</li>
		<li>Allow the agarose to set and dry overnight at room temperature.</li>
		<li>Wrap the dried slides in tissue paper and store them in a box.</li>
	</ol>
	</li>
</ol>
2. Preparation of samples
<ol>
	<li>Cultured cells 
	NOTE: Firstly, treat cells with the damaging agent(s) prior to starting the comet assay. Then, perform the following.
	<ol>
		<li>If the cells are adherent, trypsinize the cells to release them from the cell culture flask or cell culture Petri dishes, at the appropriate confluency of cells. Neutralize trypsin by adding serum-containing media.</li>
		<li>Transfer the cells to a 50 mL tube, centrifuge (e.g., for HaCaTs, centrifuge at 300 x g for 5 min at room temperature), gently remove the supernatant, and add 1 mL of PBS to the cell pellet.</li>
		<li>Perform cell counting.</li>
		<li>Transfer 30,000 cells to a 1.5 mL microcentrifuge tube and centrifuge at 7,607 x g for 5 min at 4 &#176;C.</li>
		<li>Gently remove the supernatant and store the cell pellet on ice in the dark prior to performing the comet assay. 
		NOTE: Cells should regularly be tested for Mycoplasma contamination prior to performing the comet assay to prevent, among other effects, the formation of artefactual DNA damage and altered DNA damage response, as reported elsewhere23. Centrifugation conditions may be changed, as needed, depending on the cell type used.</li>
	</ol>
	</li>
	<li>Preparation of cultured cells for repair assay
	<ol>
		<li>Culture cells in the cell culture flask or Petri dishes.</li>
		<li>Wash the cells with 1 mL of PBS twice prior to treating the cells with damaging agents (e.g., for BE-M17 cells, treat with 50 &#181;M of H2O2 for 20 min) on ice to prevent the repair from occurring during the treatment.</li>
		<li>Wash the cells gently with 1 mL of PBS twice to remove any residual damaging agents.</li>
		<li>Re-introduce the cell culture medium and allow the cells to repair for varying durations (e.g., 0 min, 30 min, 2 h, 6 h, 24 h, and 30 h) in a humidified incubator (37 &#176;C, 5% CO2).</li>
		<li>At each time point, collect 30,000 cells in 10% dimethyl sulfoxide (DMSO)-containing cell culture medium and store them at -80 &#176;C.</li>
		<li>Before performing the comet assay, thaw the cells quickly at 37 &#176;C in a water bath and centrifuge them at 7,607 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant and store the cell pellet on ice prior to performing the assay (i.e., from step 3).</li>
	</ol>
	</li>
	<li>Preparation of whole blood 
	NOTE: The following method benefits from (i) being a minimally invasive approach to obtain a blood sample, (ii) not requiring the isolation of PBMC before the comet assay, and (iii) allowing the blood samples (with a volume &#60; 250 &#181;L) to be stored at -80 &#176;C, for up to 1 month (although more recent evidence suggests that longer storage is possible), without the need for cryopreservatives, and with no risk or artefactual damage formation14. Ethical approval, or equivalent, may be needed before obtaining blood samples from patients or animals. Alternatively, commercially available blood samples can be used as in the present study. At our institution, Institutional Review Board approval is not needed for the use of commercially available blood.
	<ol>
		<li>Using a pipette, transfer whole blood samples (&#60;250 &#181;L) (Table of Materials) into a collection tube containing a minimal volume containing 0.4 mg of EDTA (per 250 &#181;L of blood).</li>
		<li>Freeze the blood samples at -80 &#176;C before performing the HTP comet assay.</li>
		<li>Thaw stored blood samples (&#60;250 &#181;L) at room temperature, without heating.</li>
		<li>Transfer 5 &#181;L of the whole blood to microcentrifuge tubes prior to performing the comet assay (see step 3).</li>
	</ol>
	</li>
</ol>
3. Cell lysis
NOTE: Carry out all the procedures on ice.
<ol>
	<li>Use 12,000 cells or 2.5 &#181;L of whole blood per gel.</li>
	<li>Prepare 0.6% (w/v) low melting point agarose dissolved in PBS using a microwave, and place in a water bath at 37 &#176;C to prevent it from solidifying.</li>
	<li>Label the frosted end of the pre-coated slides with the investigator&#39;s name, date, and treatment information using a permanent marker or pencil.</li>
	<li>Place a chilling plate on a flat bench and insert the two frozen cooling packs into the sliding drawer below the metal surface (as shown in Figure 4)21.</li>
	<li>Place the slides on the chilling plate and allow the slides to pre-chill for 1-2 min before adding the 0.6% (w/v) low melting point agarose-containing cells (step 3.7). 
	NOTE: Leaving the slides on the chilling plate for more than 1-2 min may cause condensation to form on the slide surface due to ambient humidity. This may make the low melting point agarose gels less stable on the slides.</li>
	<li>Disperse the pellet (step 2.2.7) by vortexing. Ensure that all the supernatant has been removed from the pellet. Place the sample tubes (containing the pelleted cells) immediately back on ice. 
	NOTE: When placing the sample-containing tubes in the centrifuge, put them with the hinge facing outward so that the pellet will be collected on this side of the tube. Sometimes it is difficult to see the pellet, and it is easy to dislodge it while removing the supernatant. Centrifuging with the tube lid in this orientation will enable one to know where the cell pellet will be.</li>
	<li>Resuspend the cell pellet with 200 &#181;L of 0.6% low melting point agarose (LMP agarose) and mix by pipetting up and down without creating bubbles. Next, quickly transfer 80 &#181;L of LMP agarose-containing cells onto a chilled slide and quickly place a coverslip onto the gel.</li>
	<li>Allow the gel to set on the chilling plate for 1-2 min.</li>
	<li>Meanwhile, prepare a 500 mL working solution of lysis buffer (Table 1) and pour it into the lysis dish (Figure 2).</li>
	<li>Once the gels have been set, remove the coverslips quickly by gently holding the slide between thumb and forefinger and sliding the coverslip off the gel.</li>
	<li>Place the slides containing samples inside the slide carrier (all the black &#34;dot&#34; marks on slides should be facing in the same direction when they are placed in a carrier) (Figure 3B), and then place the slide carrier inside the lysis dish (Figure 2).</li>
	<li>Close the lid of the lysis dish and keep the lysis dish in the fridge overnight at 4 &#176;C or 30 min at room temperature, whichever best suits the operator&#39;s time schedule19.</li>
</ol>
4. Electrophoresis
<ol>
	<li>Carefully remove the slide carrier from the lysis dish. Take care not to disturb the gels.</li>
	<li>Gently place the slide carrier in a washing dish pre-loaded with ice-cold ddH2O and leave it for 30 min ensuring that slides are completely covered with ddH2O.</li>
	<li>Insert a frozen cooling pack inside the sliding drawer under the electrophoresis tank to maintain optimal buffer temperature.</li>
	<li>Carefully add ice-cold electrophoresis working solution (Table 1) to the electrophoresis tank and transfer the slide carrier into the electrophoresis tank. Orientate the slides such that their clear parts with the cell-containing gels (i.e., NOT the frosted/labeling ends) point toward the cathode (red electrode).</li>
	<li>Allow the slides to sit in the electrophoresis tank for 20 min so that the DNA relaxes and unwinds. Keep the power supply swtiched off during this step.</li>
	<li>If needed, insert a new frozen cooling pack to maximize chilling.</li>
	<li>Perform electrophoresis for 20 min at 1.19 V/cm, or whatever conditions have been optimized. 
	NOTE: Optimization of electrophoresis running conditions and volume of buffer is recommended for every laboratory24. Using only a single slide carrier during electrophoresis does not cause any effect of slides on the resistance of the electrophoresis buffer, and the authors did not see a significant effect in the voltage or current when the number of the slides changed.</li>
	<li>Switch off the power supply, carefully remove the slide carrier from the electrophoresis tank, and allow it to drain on tissue paper for 30 s.</li>
	<li>Place the slide carrier into the dish containing neutralization buffer (Table 1). Leave it for 20 min.</li>
	<li>Remove the slide carrier from the neutralization dish, place it in the washing dish containing ice-cold ddH2O, and leave it for 20 min.</li>
	<li>Remove the slide carrier from the water and allow the slides to dry in an incubator at 37 &#176;C for 1 h, or at room temperature overnight, or do not dry, depending upon the operator&#39;s schedule19. 
	NOTE: If there is no drying in step 4.11, perform the staining step from 5.2.</li>
</ol>
5. Propidium iodide (PI) staining
<ol>
	<li>Transfer the slide carrier to a washing dish containing ice-cold ddH2O to rehydrate the slides and leave for 30 min.</li>
	<li>Place the slide carrier into a staining dish containing 2.5 &#181;g/mL propidium iodide solution. 
	NOTE: Propidium iodide is light-sensitive, so handle it in a darkened area. It is also toxic.</li>
	<li>Close the lid of the staining dish and incubate it for 20 min in the dark at room temperature.</li>
	<li>Transfer the slide carrier to a separate dish, and wash it with ice-cold ddH2O for 20 min.</li>
	<li>Remove the slide carrier from the dish and dry it completely in the dark, either in a 37 &#176;C incubator or at room temperature, depending upon the operator&#39;s schedule or preference.</li>
	<li>Once the slides are fully dried, remove them from the slide carrier and store them in a slide box in the dark until ready for image analysis. 
	NOTE: The slides will remain readable indefinitely and can be re-stained if necessary.</li>
</ol>
6. Enzyme-modified alkaline comet assay
NOTE: The enzyme-modified alkaline comet assay employs an enzyme treatment step after lysis but before electrophoresis. The activity of the enzyme causes breaks in the DNA at sites that are substrates for the enzyme. Before performing this assay, enzyme concentration and duration of enzyme incubation must be optimized.
<ol>
	<li>After the cell lysis (step 3), wash slides twice with ice-cold ddH2O for 20 min each.</li>
	<li>Remove the slide carrier from the water and transfer the slides to a tray lined with paper towels.</li>
	<li>Add 80 &#181;L of the enzyme at the optimized concentration (e.g., 3.2 U/mL of hOGG1 for BE-M17 cells, diluted in enzyme reaction buffer) and cover with a coverslip to spread the enzyme over the gel-containing sample.</li>
	<li>Incubate the slides at 37 &#176;C for the optimized duration (e.g., 45 min for hOGG1).</li>
	<li>After incubation, remove the coverslips gently and transfer the slides to the carrier. 
	NOTE: Do not wash the slides after enzyme treatment; directly perform electrophoresis from step 4.3.</li>
</ol>
7. DNA inter-strand crosslinks (ICL)-modified alkaline comet assay
NOTE: The concept of this variant of the ICL-ACA is that the presence of ICL in DNA will retard the electrophoretic migration of damaged DNA, induced following exposure to an oxidatively generated insult. In this instance, the shorter the comet tail, the greater the number of ICL25,26,27,28.
<ol>
	<li>Treat cells with a reagent that induces ICL (e.g., cisplatin; see Supplementary File).</li>
	<li>Expose the treated cells with one of the following agents to induce sufficient strand breaks to create a suitably sized comet tail (~20% tail DNA): hydrogen peroxide (50 &#181;M H2O2 for 30 min), ionizing radiation (2-5 Gy), or Ultraviolet B (UVB) (0.5 J/cm2).</li>
	<li>Additionally, produce a strand break positive control by treating a batch of cells with the same agent and dose, as used in step 7.2 (i.e., no treatment with ICL-inducing agent).</li>
	<li>Centrifuge the cells at 7,607 x g for 5 min, discard the supernatant, wash the cell pellet thrice with 1 mL of PBS, and process as for the alkaline comet assay (steps 3-5).</li>
	<li>Calculate levels of DNA inter-strand cross-linking using the formula below. 
	<img alt="Equation 1" src="/files/ftp_upload/63559/63559eq01.jpg" style="margin: auto;" /> 
	NOTE: MOTM (Mean Olive Tail Moment) is a comet assay endpoint widely used when describing the ICL-modified comet assay, and is defined as the product of the tail length and the fraction of total DNA in the tail (i.e., tail moment = tail length x % of DNA in the tail)29; TMdi: tail moment of samples treated with both crosslinking agent and H2O2 (or other strand breaker inducer); TMcu: tail moment of samples not treated with a crosslinking agent and not treated with H2O2 (no treatment), and TMci: tail moment of samples not treated with a crosslinking agent but treated with H2O2.</li>
</ol>
8. Comet scoring and data analysis
NOTE: The term &#34;comet&#34; derives from the images of damaged cells when viewed under a microscope after the assay has been performed (Figure 5). Under electrophoresis conditions, DNA in the undamaged cells largely does not migrate, but remains in a spheroid termed as comet &#34;head&#34;. However, the presence of strand breaks allows the cell&#39;s DNA to migrate out of the head, and form a &#34;tail&#34;, thus leading to an appearance like a comet (Figure 5). The more DNA in the tail, the more damage is present.
<ol>
	<li>Turn on the fluorescence microscope with the PI (red) filter (&#955; = 536/617 nm) and the comet assay scoring software.</li>
	<li>Add a drop of water using a Pasteur pipette to the gel and cover with a coverslip.</li>
	<li>Place the slides into the fluorescence microscope and &#34;score&#34; the comets. 
	NOTE: Scoring is a means by which the comets are assessed, to determine the amount of damage present in each comet. Broadly, this can be achieved by using two approaches, according to the user&#39;s chosen preference, either by eye (gauging the size of the comets on a scale of zero to four) or by using freely, or commercially available software30. Generally, both approaches assess the size of the comet tail, although a variety of comet-related endpoints can be determined. If using the software, click on the middle of the comet head and wait until the software detects the comet automatically, and then assesses the chosen endpoint (Figure 5).</li>
	<li>Score 50 comets per gel and 100 comets per sample (i.e., each sample corresponding to different DNA damaging treatments, or their replicates).</li>
	<li>Replicate the experiments (n = 2) or triplicate the experiments (n = 3). 
	NOTE: If only n=2 replicate experiments are undertaken, statistical analysis cannot be performed, but if n=3, perform the D&#39;agostino normality test. Most comet assay data does not pass a normality test. In this case, use a nonparametric test (Kruskal-Wallis test with Dunn&#39;s multiple comparisons test, and Mann-Whitney tests significance set at p &#60; 0.05).</li>
</ol>

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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=Comet+assay+measures+of+DNA+damage+are+predictive+of+bladder+cancer+cell+treatment+sensitivity+in+vitro+and+outcome+in+vivo.">Comet assay measures of DNA damage are predictive of bladder cancer cell treatment sensitivity in vitro and outcome in vivo.</a> International Journal of Cancer. 134 (5), 1102-1111 (2014).</li><li>Abdulwahed, A. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Investigation of DNA Damage and Genomic Organization in the Cellular Response to Platinum Chemotherapy. , (2020).</li><li>Olive, P. L., Ban&#225;th, J. P., Durand, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+in+radiation-induced+DNA+damage+and+repair+in+tumor+and+normal+cells+measured+using+the+&amp;#34;comet&amp;#34;+assay.">Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the &amp;#34;comet&amp;#34; assay.</a> Radiation Research. 122 (1), 86-94 (1990).</li><li>Kumaravel, T. S., Vilhar, B., Faux, S. P., Jha, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comet+Assay+measurements:+a+perspective.">Comet Assay measurements: a perspective.</a> Cell Biology and Toxicology. 25 (1), 53-64 (2009).</li><li>Ji, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Formation and Repair of Environmetally-induced damage to Mitochondrial and Nuclear Genomess. , (2020).</li></ol>

Dr. Cooke and Dr. Karbaschi are inventors on three granted patents relating to the technologies described here.

This study demonstrates the versatility provided by the current equipment, which can be used to achieve high throughput with a variety of representative, common variants of the comet assay (i.e., alkaline, enzyme-modified, blood, and ICL, and other variants will be suitable too). In addition, the present approach brings with it several benefits20,21: (a) assay run time is decreased due to manipulation of multiple slides in parallel (handling time decreases by 60%); (b) risk of damage to gels, and hence the risk to the experiment are decreased; (c) reagent requirements are decreased (e.g., the volume of the electrophoresis tank is smaller than the conventional tank); (d) the number of slides run is increased. One tank can provide a 20% increase in the number of slides run compared to a single conventional tank; however, multiple electrophoresis tanks can be run or slaved (i.e., multiple tanks controlled by a single power supply), in parallel from the same power supply, and still require a benchtop footprint smaller than a single conventional tank with ice tray; and (e) tank footprint is decreased due to vertical orientation of slides and integral cooling (saves lab space); the HTP tank comprises a high-performance ceramic cooling base with a sliding drawer that can fit one frozen cooling pack to maintain optimal buffer temperature without having to perform the process in a cold room.
Moreover, the chilling plate developed by us accommodates 26 comet slides, enables rapid solidification of the low melting point agarose on the comet assay slides and facilitates an easy retrieval of the slides after the agarose gel is solidified. The above innovations make the comet assay process simpler and easier.
While other high-throughput approaches have been developed (e.g., 12-gel comet assay, CometChip, or 96 mini-gel formats)25, many scientists prefer using the conventional microscope slides (which includes the commercially available pre-coated slides, or other specialized slides). The present approach can accommodate all types of microscope slides, allowing experiments using these slides to be scaled up through faster slide processing and handling. As noted above, the HTP comet system brings many advantages, but there is one notable limitation: the current approach provides only a 20% increase in the number of samples run, compared to a conventional horizontal tank (although processing of slides is much faster). The CometChip and 96 mini-gel formats run a greater number of samples. To date, we do not know whether the present approach can accommodate the CometChip or 96 mini-gel formats, although we predict that it will. As noted above, the number of samples can be increased further by slaving tanks to a single power supply. As with all approaches, there is still a chance of losing or damaging the gels while loading samples and analyzing them under the microscope, but this is more due to operator error, and the chances of this are minimized with the current approach.
The use of the HTP comet system can greatly help analyze DNA damage, facilitating the use of the comet assay in a wide range of applications, such as molecular epidemiology, male reproductive science, genotoxicology studies, and environmental toxicology. This is particularly true for those users who wish to have all the benefits of improved throughput, and ease of use, without moving away from the familiar, cost-effective, conventional microscope slides.

Cells are exposed continually to agents arising from the internal and external environments, which can damage DNA1,2. This damage can cause aberrant cell function3, and therefore DNA damage may play a critical role in the development of many major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging4. The comet assay (also called single-cell gel electrophoresis) is an increasingly popular method for detecting and quantifying cellular DNA damage.
At its simplest, the alkaline comet assay (ACA) detects strand breaks (SB; both single and double), together with apurinic/apyrimidinic sites and alkali-labile sites (ALS) both of which become single-strand breaks under alkaline conditions5. The neutral pH comet assay can evaluate frank single- and double-strand breaks6. Furthermore, the ACA, in combination with a number of DNA repair enzymes, can detect a considerable range of types of DNA damage, e.g., oxidized purines (identified by the use of human 8-oxoguanine DNA glycosylase 1; hOGG17); oxidized pyrimidines (using Endonuclease III; EndoIII) and cyclobutane pyrimidine dimers (using T4 endonuclease V; T4endoV)8. The comet assay can also be used to evaluate DNA lesions induced by crosslinking agents, such as cisplatin9,10,11. As indicated by the assay&#39;s formal name, i.e., single cell gel electrophoresis, the assay relies upon the cells under analysis being a single cell suspension; most commonly, these are cultured cells but may be isolated from whole blood12,13, or whole blood itself can be used14,15. Alternatively, a single cell suspension may be generated from solid tissues.
Apart from a few exceptions, most notably the CometChip reports from the Engleward lab16, the overall comet assay protocol has not changed dramatically from that originally described by the assay&#39;s inventors (&#214;stling and Johansson17 and Singh et al.18). The comet assay involves numerous steps (Figure 1). Many of these steps involve the transfer of the thin, cell-containing agarose gels, one slide at a time, and, therefore, pose a risk of damage or loss of the gel, jeopardizing the experiment&#39;s success. Consequently, the comet assay can be time-consuming, particularly if a significant number of slides are being run. Typically, a maximum of 40 slides are run in a large (33 cm x 59 cm x 9 cm) electrophoresis tank, which sits within an even larger tray containing wet ice for cooling. It has been recently reported that the assay runtime can be shortened to 1 day by decreasing the duration of the lysis step and not drying the slides before staining19.
The present authors have previously reported a novel approach to the high throughput alkaline comet assay (HTP ACA), in which multiple (batches of 25) comet assay microscope slides can be manipulated simultaneously throughout the comet assay process20,21,22. This patented approach minimizes the risk of damage to, or loss of, the sample-containing gels by removing the need to manipulate the microscope slides individually and can be applied to all variants of the comet assay, which use microscope slides. The slide-containing racks protect the gels during the manipulations, and consequently, the sample processing is quicker and more efficient. The slides can also undergo electrophoresis in the racks, held in the vertical, rather than horizontal, orientation. This, and integral cooling, significantly decrease the footprint of the electrophoresis tank and removes the need for wet ice. Taken together, this represents a significant improvement over the conventional procedure. The equipment used is illustrated in Figure 2. The protocols described here, using this novel approach, demonstrate the representative application to cultured cells and whole blood14 for detection of alkali-labile sites (ALS), DNA inter-strand crosslinks (ICL), and the substrates of various DNA repair enzymes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>22 x 22 mm glass coverslips</td>
			<td>Fisher Scientific, Hampton, NH, USA</td>
			<td>631-0124</td>
			<td></td>
		</tr>
		<tr>
			<td>A2780</td>
			<td>ECACC,&#160; 
			Louis, MO, 
			&#160;USA</td>
			<td>93112519</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrated nitric acid (OptimaTM grade)</td>
			<td>Fisher Scientific Fair Lawn, NJ, USA</td>
			<td>A467-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope equipped with a camera</td>
			<td>Zeiss, Jena, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fresh human whole blood</td>
			<td>Zen Bio Inc</td>
			<td>SER-WB10ML</td>
			<td>Commercial human whole blood sample</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad Software, San Diego, California</td>
			<td></td>
			<td>Data analysis software</td>
		</tr>
		<tr>
			<td>HTP Comet Assay system</td>
			<td>Cleaver Scientific</td>
			<td>COMPAC-&#160;50</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Keratinocyte (HaCaTs)</td>
			<td>American Type Culture Collection (ATCC), Manassas, VA, USA</td>
			<td>Discontinued</td>
			<td>Can be purchased from another company 
			ADDEXBIO TECHNOLOGIES 
			Cat# T0020001</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2) 
			30% in water</td>
			<td>Fisher Scientific, Hampton, NH, USA</td>
			<td>BP2633-500</td>
			<td></td>
		</tr>
		<tr>
			<td>ICP-MS 
			iCAP RQ ICP-MS system</td>
			<td>Thermo Scientific, 
			Waltham, MA, 
			USA</td>
			<td>IQLAAGGAAQFAQKMBIT</td>
			<td></td>
		</tr>
		<tr>
			<td>Image and Data Analysis software</td>
			<td>Perceptive Instrument, 
			Bury St Edmunds, England, 
			UK</td>
			<td>125525</td>
			<td>Free image analysis softwared is available e.g., ImageJ</td>
		</tr>
		<tr>
			<td>Internal Standard Mix</td>
			<td>SPEX Certiprep, 
			Metuchen, NJ, USA</td>
			<td>CL-ISM1-500</td>
			<td>Bismuch (isotope monitored 209 Bi)-concnetration of 10 &#181;g/mL in 5% HNO3</td>
		</tr>
		<tr>
			<td>Low melting point Agarose</td>
			<td>Invitrogen 
			Waltham, MA, USA</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2EDTA (disodium ethylenediaminetetraacetic acid)</td>
			<td>Sigma Aldrich, 
			St. Louis, MO, 
			USA</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (Sodium chloride)</td>
			<td>Sigma Aldrich, 
			St. Louis, MO, 
			USA</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>Thermo Scientific, 
			Waltham, MA, 
			USA</td>
			<td>701-058108</td>
			<td>Nanodrop for measuring DNA concentration</td>
		</tr>
		<tr>
			<td>Nanopure Infinity Ultrapure Water System (Barnstead Nanopure)</td>
			<td>Thermo Scientific,&#160; 
			Waltham, MA, 
			USA</td>
			<td>D11901</td>
			<td>Ultrapure water (16 M&#937; cm-1)</td>
		</tr>
		<tr>
			<td>NaOH (sodium Hydroxide)</td>
			<td>Sigma Aldrich, 
			St. Louis, MO, USA</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal melting point Agarose</td>
			<td>Fisher Scientific, 
			Hampton, NH, USA</td>
			<td>16520100</td>
			<td>For pre-coating slides</td>
		</tr>
		<tr>
			<td>OCI-P5X</td>
			<td>University of Miami, 
			Miami, FL, 
			USA</td>
			<td>N/A</td>
			<td>Live Tumor Culture Core facility provided the cells</td>
		</tr>
		<tr>
			<td>Platinum (Pt) reference standard</td>
			<td>SPEX Certiprep, 
			Metuchen, NJ, USA</td>
			<td>PLPT3-2Y</td>
			<td>(1000 &#181;g/mL in 10% HCl) containing Bismuch</td>
		</tr>
		<tr>
			<td>Propidium Iodide 
			(1.0 mg/mL in water)</td>
			<td>Sigma Aldrich, 
			St. Louis, MO, 
			USA</td>
			<td>12-541BP486410ML</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit</td>
			<td>Qiagen 
			Valencia, CA, 
			USA</td>
			<td>51304</td>
			<td>DNA extraction Kit</td>
		</tr>
		<tr>
			<td>Single-frosted glass microscope slides</td>
			<td>Fisher Scientific, 
			Hampton, NH, USA</td>
			<td>12-541B</td>
			<td></td>
		</tr>
		<tr>
			<td>SKOV3</td>
			<td>ECACC, 
			Louis, MO, 
			USA</td>
			<td>91091004</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide box</td>
			<td>Fisher Scientific, 
			Hampton, NH, USA</td>
			<td>03-448-2</td>
			<td>Light proof, to protect cells from the formation adventitious damage (according to the widely held view) and prevent fading of the fluorescent dye</td>
		</tr>
		<tr>
			<td>Slide Chilling plate</td>
			<td>Cleaver Scientific, 
			Rugby, England, 
			UK</td>
			<td>CSL-CHILLPLATE</td>
			<td></td>
		</tr>
		<tr>
			<td>Treatment dish</td>
			<td>Cleaver Scientific, 
			Rugby, England, 
			UK</td>
			<td>STAINDISH4X</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Sigma Aldrich, 
			St. Louis, MO, 
			USA</td>
			<td>93362</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific, 
			Hampton, NH, USA</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA (0.5%)</td>
			<td>Invitrogen 
			Gibco, 
			Waltham, MA, USA</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical Slide Carrier</td>
			<td>Cleaver Scientific, 
			Rugby, England, 
			UK</td>
			<td>COMPAC-25</td>
			<td></td>
		</tr>
	</tbody>
</table>

a high-throughput comet assay approach for assessing cellular dna damage

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

Optimization of the electrophoresis voltage for the HTP ACA 
Human keratinocytes (HaCaTs; Table of Materials) were irradiated with different doses of ultraviolet A radiation (UVA) (5 or 10 J/cm2; Figure 6A), UVB (0.5 or 1 J/cm2; Figure 6B), or treated with 50 &#181;M H2O2 (Figure 6C) to induce damage. Three different voltages of the electrophoresis were tested to determine the optimal voltage for electrophoresis. The results from all three DNA damaging treatments revealed that, while all voltages generated linear dose-responses, the most sensitive response was obtained with 1.19 V/cm. HaCaTs showed the highest baseline DNA damage using 1.19 V/cm during electrophoresis compared to 1 V/cm and 1.09 V/cm (Figure 6A-C). In addition, using 1.19 V/cm, the greatest % tail DNA is seen, following all damaging treatments (Figure 6)31.
Detection of DNA damage in human whole blood using Fpg modified HTP ACA 
Human whold blood (Table of Materials) was irradiated with different doses of 10 J/cm2 UVA to induce damage. Four different concentrations of Fpg (1, 2, 4 or 8 U/mL) were used to determine the optimal concentration for enzyme treatment in HTP ACA. The results showed that the optimal levels of DNA damage were revealed with 4 U/mL Fpg (Figure 7A). Representative comet images from UVA irradiated blood samples (Figure 7B).
Detection of DNA ICL in a representative ovarian cancer cell line using the ICL-modified HTP ACA 
An ovarian cancer cell line (SKOV-3; Table of Materials) was treated with combinations of 200 &#181;M cisplatin and/or subsequent treatment with 50 &#181;M H2O2 for 30 min on ice. No appreciable damage was noted in the unexposed cells (Figure 8A). Exposure to H2O2 alone generated a significant MOTM (Figure 8B). In contrast, the cells in which ICL were induced showed a decreased MOTM (Figure 8C)28.
Formation and repair of cisplatin-induced DNA ICL in a representative, ovarian cancer cell line 
The ICL-modified HTP ACA was used to determine the time course for DNA ICL formation and repair induced by cisplatin in an ovarian cancer cell line (A2780; Table of Materials). The cells were treated with 100 &#181;M cisplatin for 1 h, and then incubated in cisplatin-free media (RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS)) for a subsequent time course. At various time points, the ICL-modified HTP ACA was performed to establish the levels of ICL (Figure 9)28. No ICL were detected prior to cisplatin treatment. However, after a single treatment with 100 &#181;M cisplatin, ICL levels increased significantly, peaking at 12 h, after which levels decreased back to zero after 30 h.
Correlation between DNA ICL and DNA platinum levels 
Three ovarian cancer cells were treated with 100 &#181;M cisplatin to induce different levels of DNA-ICL, prior to analysis by the ICL-modified HTP ACA and inductively-coupled mass spectrometry (ICP-MS; see Supplementary File for details). As shown in Figure 10, differing levels of DNA-ICL were induced in the three cell lines, together with differing levels of Pt in DNA. A positive correlation (R2 = 0.9235) was observed between DNA ICL levels and platinum concentrations, indicating the association between DNA platinum levels and the corresponding ICL28.
Base excision repair in Mycoplasma-infected and uninfected BE-M17 cells 
Mycoplasma infected and uninfected BE-M17 cells were treated with 50 &#181;M H2O2 for 30 min and incubated with complete medium (Dulbecco&#39;s modified Eagle&#39;s medium supplemented with 10% (v/v) FBS) for different durations (0 min, 30 min, 1 h, 2 h, 6 h, 24 h, or 30 h) during which cells were allowed to repair. At each time point, cells were collected and frozen at -80 &#730;C, in a 10% DMSO-containing medium, before performing the hOGG1-modified HTP ACA (step 6). After 30 min, levels of SB/ALS had decreased to 21% TD (percentage tail DNA) in the uninfected cells, whereas the infected cells showed 49% TD (Figure 11A). After ~15 h, levels of SB/ALS had returned to baseline in both infected and uninfected cells. For the oxidized purines, the uninfected BE-M17 initially showed a small increase in damage, before returning to baseline within 30 h (Figure 11B). In contrast, the infected cells showed a sustained, significant increase in oxidized purines, which remained elevated, and did not return to baseline levels even after 30 h (Figure 11B)23.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63559/63559fig01.jpg" /> 
Figure 1: Overview of the conventional alkaline comet assay procedure. (i) A single-cell suspension of cultured cells or a sample of whole blood is mixed with 0.6% (w/v) LMP agarose. (ii) The cell/agarose mixture is applied to pre-coated microscope slides and covered with coverslip until solidified. (iii) The cells are lysed using a high pH lysis buffer overnight, forming nucleoid bodies, before (iv) washing with ddH2O. (v) The cellular DNA unwinds in the high pH electrophoresis buffer. The presence of strand breaks allows the DNA to relax and unwind, and under electrophoresis, the DNA is drawn out of the nucleoid body, forming a tail. The slides are then (vi) drained, dried, (vii) neutralized, and (viii) washed with ddH2O before (ix) being dried overnight. Slides are then (x) rehydrated with ddH2O, (xi) stained, (xii) washed, and finally (xiii) scored and analyzed, typically using fluorescent microscopy and image analysis software. This figure is reproduced from a previous publication20. <a href="https://www.jove.com/files/ftp_upload/63559/63559fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63559/63559fig02.jpg" /> 
Figure 2: The materials comprising the high-throughput comet electrophoresis system. HTP electrophoresis tank, HTP racks, and the dishes for lysis, wash, neutralization, and staining are shown. <a href="https://www.jove.com/files/ftp_upload/63559/63559fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63559/63559fig03.jpg" /> 
Figure 3: Representative images of a comet assay slide and HTP rack (microscope slide carrier). (A) For correct orientation, the pre-coated face of the microscope slide is recognized by a black dot in the right-hand corner of a microscope slide. (B) The image of the HTP rack illustrates how the slides are kept in a tight vertical orientation, with tabs on the carrier to fix its orientation within the electrophoresis tank. Each carrier can accommodate up to 25 slides. <a href="https://www.jove.com/files/ftp_upload/63559/63559fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63559/63559fig04.jpg" /> 
Figure 4: Representation of the chilling plate with sample slides and freezer packs in place. <a href="https://www.jove.com/files/ftp_upload/63559/63559fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63559/63559fig05.jpg" /> 
Figure 5: Screenshot of representative comets taken during scoring. HaCaTs (A) without treatment and (B) treated with 1 J/cm2 UVB prior to performing HTP ACA. Most software packages can calculate a variety of comet endpoints, but the most common ones are the % tail DNA (preferred) or tail moment based upon these images (blue: start of the head, green: middle of the head, and purple: end of tail). The scale bar is 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63559/63559fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63559/63559fig06.jpg" /> 
Figure 6: Representative graphs illustrating the effect of electrophoresis voltage on percentage tail DNA, determined using the HTP ACA. Cells were exposed to (A) 5 or 10 J/cm2 UVA, (B) 0.5 or 1.0 J/cm2 UVB, or (C) 50 &#181;M H2O2 prior to the HTP ACA, with the electrophoresis voltage at either 1, 1.09, or 1.19 V/cm. Data represent the mean of 200 determinations from n = 2 duplicate experiments31. <a href="https://www.jove.com/files/ftp_upload/63559/63559fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63559/63559fig07.jpg" /> 
Figure 7: Representative graph and comet images of human blood analysed by the Fpg modified HTP ACA. Human blood samples were irradiated with 10 J/cm2 UVA or sham irradiated (&#39;ctrl&#39;) on ice prior to the lysis step. Different concentrations of Fpg (1, 2, 4, or 8 U/mL) were used for the enzyme treatment prior to electrophoresis. (A) Data represent the mean &#177; SEM of 300 determinations from n=3 experiments. (B) Representative images of comets for each concentration of Fpg in 10 J/cm2 UVA irradiated blood samples. The scale bar is 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63559/63559fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63559/63559fig08.jpg" /> 
Figure 8: Representative comet images illustrating ICL detection following cisplatin treatment. (A) Control cells without any treatment, (B) cells which were treated with H2O2 (50 &#181;M) only, (C) cells which were treated with H2O2 (50 &#181;M) and cisplatin (200 &#181;M), illustrating the tail to be shorter than in (B), due to the presence of ICL28. The scale bar is 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63559/63559fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/63559/63559fig09.jpg" /> 
Figure 9: Demonstration of the kinetics of cisplatin-induced ICL formation and repair. A2780 cells were treated with 100 &#181;M of cisplatin in the culture medium for 1 h. The cisplatin-containing medium was then removed, and the cells were cultured for various time points, before analysis by ICL-modified HTP ACA. Data represent mean &#177; SEM from n = 3 experiments28. **** P &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/63559/63559fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/63559/63559fig10v2.jpg" /> 
Figure 10: Correlation between DNA ICL and platinum concentration. DNA ICL were determined by the ICL-modified HTP ACA and platinum levels were measured by ICP-MS (with Single Quad-Kinetic Energy Discrimination, SQ-KED), in three ovarian cancer cell lines. R2 = 0.9235. See Supplementary File for ICP-MS methodology to quantify platinum levels in DNA28. <a href="https://www.jove.com/files/ftp_upload/63559/63559fig10v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/63559/63559fig11.jpg" /> 
Figure 11: A representative graph illustrating DNA damage and repair, determined by the hOGG1-modified comet assay, in Mycoplasma infected versus uninfected BE-M17 cells. After treatment with 50 &#181;M H2O2 for 30 min, cells were allowed to repair for different durations (0, 30 min, 1 h, 2 h, 6 h, 24 h, or 30 h). The hOGG1-modified HTP ACA was used to measure (A) SB/ALS and (B) oxidized purines in infected (red data points) and uninfected (black data points) BE-M17 cells. Data represent the mean of 200 determinations from n = 2 duplicate experiments. This figure is reproduced with permission from a previous publication23. <a href="https://www.jove.com/files/ftp_upload/63559/63559fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td colspan="2">Stock Solution</td>
			<td>Working solution</td>
		</tr>
		<tr>
			<td>Lysis buffer</td>
			<td colspan="2">100 mM Na2EDTA, 2.5 M NaCl, and 10 mM Tris Base in ddH2O; adjust pH to 10 with 10 M NaOH</td>
			<td>1% Triton X-100 in lysis stock solution</td>
		</tr>
		<tr>
			<td>Electrophoresis buffer</td>
			<td colspan="2">10 M NaOH and 200 mM Na2EDTA in ddH2O</td>
			<td>300 mM NaOH and 1 mM Na2EDTA; pH &#62; 13</td>
		</tr>
		<tr>
			<td>Neutralization buffer</td>
			<td colspan="2">0.4 M Tris Base in ddH2O; adjust pH to 7.5 with HCl</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining buffer</td>
			<td colspan="2">1 mg/mL propidium iodide</td>
			<td>2.5 &#181;g/mL propidium iodide in ddH2O</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of reagents used in HTP ACA. The stock and working concentrations of lysis, electrophoresis, neutralization, and staining buffers are shown.
Supplementary File. <a href="https://www.jove.com/files/ftp_upload/63559/Supplementary information.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage at JoVE.com

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell ...

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4.5K Views.  University of South Florida. The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

Video: A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...