This work was supported by an NIH grant R01DK119183 (to G.A. and M.D.C.), a pilot project award through P30DK079307 (to M.G.D.), an American Urology Association Career Development award and a Winters Foundation grant (to N.M.), and by the Cell Physiology and Model Organisms Kidney Imaging Cores of the Pittsburgh Center for Kidney Research (P30DK079307).

<ol>
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The authors have nothing to disclose.

The incorporation of video-monitoring is a cost-effective modification that presents several advantages over the classical VSA. In the classical VSA, which is typically used as an end-point assay, it is difficult to distinguish overlapping void spots. This is not a trivial concern, as mice tend to urinate multiple times in the same area when the assay is prolonged for several hours, typically in the corners of their cage. Thus, the first advantage of RT-VSA is that the investigator can readily identify individual spots that have been deposited partially or totally on top of each other. This is well illustrated in the experiment shown in Figure 12. In this case, video recording confirms that the mouse voided three times in the same corner, depositing overlapping void spots. If the filter paper was analyzed only at the end of the experiment, in the manner typical of a VSA, only one spot could be distinguished. So, while the classical, endpoint VSA can be useful in identifying large differences in voiding behavior, it may fail to discriminate between more subtle phenotypes. With the current advances in genetics and the generation of transgenic animals, the development of methods that can accurately assess voiding behavior is essential.
Second, RT-VSA provides the investigator with temporal information about the voiding events. Thus, one can determine true micturition frequencies, as well as establish relationships between voiding events. For example, in a situation where a cluster of SVSs is observed next to a single large PVS, in the classical VSA, one cannot readily discriminate if the mouse voided several times during the experiment (with three small and one large void) or if the animal deposited one large urine volume, and the three smaller spots are a result of post-micturition dribbling or carryover from fur. RT-VSA can be used to address these questions.
Third, RT-VSA allows one to analyze the behavior of a mouse before, during, and after the voiding event. The investigator can also monitor the mouse&#8217;s overall activity profile by analyzing RT-VSA recordings with freely available software such as Mouse Behavior Tracker39. In addition, one can discriminate whether an animal stops to urinate (typical behavior of females), whether the animal is in motion during voiding (which in males can result in the trailing of void spots), or whether it voids during its sleep/rest phase. We have observed the latter behavior, although it is normally rare. Thus, if properly validated and interpreted, observing voiding behavior can provide the investigator with valuable insights into voiding dysfunction, including, for example, nocturia or incontinence. RT-VSA (and VSA) allows investigators to determine the fraction of void events that occur in the central area of a cage, as opposed to the corners. Normally, center voiding is rare, and increased numbers of such events is a sign of bladder dysfunction8,22. The one exception is dominant alpha-males, which urinate frequently and across the entirety of their enclosures20. In order to perform an analysis of central voiding using RT-VSA, one should consider moving the food dish to a side wall and remove the dome (or fix it to a particular region of the cage), so spatial patterns of urination could be more readily established.
Fourth, the RT-VSA allows one to more effectively manage the stresses associated with cage exchange compared to classical VSA. Mice are skittish by nature and handling them or transferring them to a new environment causes stress, which is known to impact voiding behavior35. By its nature, it is difficult to incorporate a period of acclimatization into the standard VSA. In contrast, in RT-VSA, a period of acclimatization can be easily incorporated in each experiment, and any voiding events during this period disregarded during the analysis. An additional stress-relieving aspect of RT-VSA implemented in this protocol is the duplication of conditions under which mice are normally housed (i.e., access to food and water ad libitum, a dome, and a toy). This is, to the best of our knowledge, one of the richest environments described for this type of experiment. In a typical endpoint VSA, mice are deprived from water (and sometimes also from food)8,22, which we provide ad libitum.
Fifth, in addition to the positive effect that this enriched environment may have on the stress levels of mice, it allows the investigator to perform RT-VSA for extended periods of time, typically in 6 h time windows; however, more recently we have been performing experiments that run for 24 h (data not shown). This is ideal when assessing the effects of circadian rhythmicity or when studying mice that may exhibit an underactive bladder phenotype, and thus urinate infrequently.
For 24 h video monitoring, we recommend using thin filter paper and analyzing the experiments using the bottom cameras. This combination of type of paper and camera is the best option that allows for sensitivity of detection in the dark and light phases. As mentioned earlier, bottom cameras cannot readily detect void spots on the thick filter paper and top cameras do not reliably detect void spots during the light phase. If performing these extended analyses, we suggest placing the animals in the recording cages at 05:00 p.m., analyzing from 06:00 p.m. on day 1 and completing the experiment at 06:00 p.m. on the following day.
While RT-VSA has many positives aspects, there are some caveats worth noting. While we make every attempt to limit stress when performing these analyses, we cannot completely replicate the mouse&#8217;s habitat, as the animals are normally group-housed. They also drink water from a bottle instead of getting water from non-wetting water gel. However, our observation that mice have fewer voids during their inactive light phase versus their active dark phase indicates to us that the mice retain their normal patterns of circadian voiding behavior40. Reflecting their easily stressed nature, we found that mice exhibit disrupted voiding behavior on the days of cage cleaning. Thus, one must limit analysis to periods of time when the animals will be less impacted by routine animal husbandry. An important factor to consider is whether the UV light has any impact on voiding function. Because our findings show a distinct circadian difference in VSA parameters between light and dark phases, which are consistent with other techniques that descrive a similar phenomenon but not using UV light40, we conclude that UV light illumination does not impact voiding function. Notice that the filter paper prevents light from passing from the lower chamber to the upper chamber.
An additional caveat is that the longer one keeps the mice in the chamber, the more likely they are to chew or damage the filter paper (irrespective of the thickness of the paper). While none of the animals disrupted the paper during the light phase, almost 30% of mice damaged the paper during the dark active phase. In the analysis of the 24 h experiments, we observed that a similar proportion (one third) of the experiments could not be analyzed due to paper damage, meaning that not only the amount of time, but also the phase of the day affects the shredding behavior of the animal. Unfortunately, in our experience, we have&#160;noticed that an animal that disrupts the cage&#8217;s paper will continue to do so even upon re-testing. As noted above, mice sometimes urinate on the bare acrylic floor, and thus loss of the paper makes the analysis incomplete and of limited value.
Further, the analysis of male voiding behavior can be more difficult as they tend to void more frequently, they can dribble after voiding, and there is a population of males (20%) with a more aggressive alpha-male phenotype characterized by large numbers of small voids that are distributed across the cage, as discussed in an earlier publication3. As this alpha-male behavior can constitute a confounding factor for establishing voiding phenotypes, we exclude animals with &#8805;50 (light phase experiments) or &#8805;100 (dark phase experiments) voids from analysis.
There are situations in which the discrimination of voids based on their volume (i.e, PVS vs SVS) is not necessarily warranted. For instance, in our experience, mice with bacterial or chemical cystitis tend to void smaller volumes than controls4,34. However, there are great differences in the voided volume between individual mice; in some, most voids have a volume &#8804;20 &#956;L, while in others the majority is &#62;20 &#956;L. Consequently, the discrimination of the voided volume does not provide any advantage for the characterization of these phenotypes.
In sum, RT-VSA is an easy to implement tool for analyzing mouse voiding behavior in freely mobile mice. Unlike tools such as cystometry, it does not require surgical implantation of a catheter or non-physiological rates of bladder filling. It allows for the determination of voiding behavior in both sexes of mice, over extended periods of time, and during the light and dark phases of the day cycle. It is also relatively affordable, especially when compared to more specialized and expensive devices such as metabolic cages. While there are caveats associated with this tool, they are generally easy to manage. Finally, this technique could be easily adapted to other animal species, including other rodents.

Urine storage and micturition are coordinated by a central circuitry (central nervous system) that receives information about the bladder filling status through the pelvic and hypogastric nerves. The urothelium, the epithelium that lines the urinary tract from the renal pelvis to the proximal urethra, forms a tight barrier to the metabolic waste products and pathogens present in urine. It is an integral component of a sensory web, which senses and communicates the filling state of the bladder to underlying tissues and afferent nerves1,2. Disruption of the urothelial barrier, or alterations in urothelial mechanotransduction pathways, can lead to voiding dysfunction along with lower urinary tract symptoms such as frequency, urgency, nocturia, and incontinence3,4,5,6,7. Likewise, aging, diabetes, lower urinary tract infections, interstitial cystitis, and other disease processes that affect the urinary bladder, or the associated circuitry that controls its function, are known to cause bladder dysfunction8,9,10,11,12,13,14,15,16,17,18,19. A better understanding of normal and abnormal voiding behavior depends on the development of methods that can reliably discriminate among different urination patterns.&#160;
Traditionally, the voluntary voiding behavior of mice has been studied using the void spot assay (VSA), developed by Desjardins and colleagues20, and broadly adopted due to its simplicity, low cost, and noninvasive approach8,21,22,23,24. This assay is typically performed as an endpoint assay, in which a mouse spends a defined amount of time in a cage lined by a filter paper, which is subsequently analyzed by counting the number and assessing the size of urine spots when the filter paper is placed under ultraviolet (UV) light (the urine spots fluoresce under these conditions)20. Despite these many advantages, the traditional VSA presents some major limitations. Because mice often urinate in the same areas, investigators have to restrict the duration of the assay to a relatively short period of time (&#8804;4 h)25. Even when the VSA is performed over shorter time periods, it is almost impossible to resolve small void spots (SVSs) that fall over large void spots or, to discriminate SVSs from the carryover of urine adhered to tails or paws. It is also very difficult to distinguish if SVSs are a consequence of frequent but individual voiding events (a phenotype that is often observed in response to cystitis4,26), or due to post-micturition dribbling (a phenotype associated with bladder outlet obstruction27). Furthermore, the desire to complete the assay during working hours, coupled with difficulties accessing housing facilities when the lights are turned off, often limits these assays to the light period of the 24 h circadian cycle. Thus, these time constraints prevent the evaluation of mouse voiding behavior during their active night phase, lessening the ability to analyze specific genes or treatments that are governed by circadian rhythms.&#160;
To overcome some of these limitations, researchers have developed alternative methods to assess voiding behavior in real time26,28,29,30,31,32. Some of these approaches involve the use of expensive equipment such as metabolic cages26,28,29, or the use of thermal cameras30; however, these too have limitations. For example, in metabolic cages, urine tends to adhere to the wires of the mesh floor and to the walls of the funnel, reducing the amount of urine that is collected and measured. Thus, it can be difficult to accurately collect data about small voids. Moreover, metabolic cages do not provide information about the spatial distribution of the voiding events (i.e., urination in the corners vs. the center of the chamber). Given that long-wavelength infrared radiation used by the thermographic cameras does not penetrate solids, voiding activity assessed by video thermography must be performed in an open system, which can be challenging with active mice, as they can jump several inches in the air. Another system is the automated voided stain on paper (aVSOP) approach33, which consists of rolled filter paper that winds up at a constant speed below the wire mesh floor of a mouse cage. This approach prevents paper damage and the overlap of urine spots that occur in the classical VSA, and its implementation allows the investigator to perform experiments over several days. However, it does not provide the investigator with precise timing of the voiding events, and there is no ability to examine behavior and how it correlates with spotting. To obtain this information, researchers have incorporated video-monitoring to voiding assays, an approach that allows the simultaneous assessment of mouse activity and urination events31,32. One approach consists of placing a blue light emitting diode (LED) and a video-camera with a green fluorescence protein filter set under the experimental cage to visualize the voiding events, and an infrared LED and a video-camera above the cage to capture mouse position32. This setup has been used to monitor voiding behavior while performing fiber photometry; however, the brightly lit environment of this system required the investigators to treat their mice with a diuretic agent to stimulate voiding. In another experimental design, wide-angle cameras were placed above and below the experimental cage to visualize mouse motor activity and urination events, respectively. In this case, urine spots deposited on a filter paper lining the cage&#8217;s floor were revealed by illuminating the filter paper with UV lights placed under the cage31. This setup was used in short assays, 4 min in duration, during the light phase of the day to study the brainstem neurons involved in voluntary voiding behavior31. The suitability of this system for its use during the dark phase or for periods of time &#62;4 min was not reported.&#160;
In this article, a method is described that enhances the traditional VSA by allowing for long-term video monitoring of mouse voiding behavior. This cost-effective approach provides temporal, spatial, and volumetric information about voiding events for extended periods of time during the light and dark phases of the day, along with details related to mouse behavior3,4,34. Detailed information for the construction of the voiding chambers, the implementation of a real-time VSA (RT-VSA), and the analysis of the data is provided. The RT-VSA is valuable for researchers seeking to understand the physiological mechanisms that control the function of the urinary system, to develop pharmacological approaches to control micturition, and to define the molecular basis of disease processes that affect the lower urinary tract.&#160;

<table><tbody><tr><td>1.00&amp;rdquo; X 1.00&amp;rdquo; T-Slotted Profile - Four Open T-Slots &amp;ndash;&amp;nbsp; cut to 10 inches</td><td>80/20</td><td>1010</td><td>Amount: 20</td></tr><tr><td>1.00&amp;rdquo; X 1.00&amp;rdquo; T-Slotted Profile - Four Open T-Slots &amp;ndash;&amp;nbsp; cut to 12 inches</td><td>80/20</td><td>1010</td><td>Amount: 6</td></tr><tr><td>1.00&amp;rdquo; X 1.00&amp;rdquo; T-Slotted Profile - Four Open T-Slots &amp;ndash;&amp;nbsp; cut to 40 inches</td><td>80/20</td><td>1010</td><td>Amount: 4</td></tr><tr><td>1.00&amp;rdquo; X 1.00&amp;rdquo; T-Slotted Profile - Four Open T-Slots &amp;ndash; cut to 14.75 inches</td><td>80/20</td><td>1010</td><td>Amount: 12</td></tr><tr><td>1.00&amp;rdquo; X 1.00&amp;rdquo; T-Slotted Profile - Four Open T-Slots &amp;ndash; cut to 32 inches</td><td>80/20</td><td>1010</td><td>Amount: 5</td></tr><tr><td>1/4-20 Double Slide-in Economy T-Nut</td><td>80/20</td><td>3280</td><td>Amount: 16</td></tr><tr><td>1/4-20 Triple Slide-in Economy T-Nut</td><td>80/20</td><td>3287</td><td>Amount: 18</td></tr><tr><td>10 &amp;amp; 25 Series 2 Hole - 18mm Slotted Inside Corner Bracket with Dual Support</td><td>80/20</td><td>14061</td><td>Amount: 6</td></tr><tr><td>10 Series 3 Hole - Straight Flat Plate</td><td>80/20</td><td>4118</td><td>Amount: 8</td></tr><tr><td>10 Series 5 Hole - &quot;L&quot; Flat Plate</td><td>80/20</td><td>4081</td><td>Amount: 8</td></tr><tr><td>10 Series 5 Hole - &quot;T&quot; Flat Plate</td><td>80/20</td><td>4080</td><td>Amount: 8</td></tr><tr><td>10 Series 5 Hole - Tee Flat Plate</td><td>80/20</td><td>4140</td><td>Amount: 2</td></tr><tr><td>10 Series Standard Lift-Off Hinge - Right Hand Assembly</td><td>80/20</td><td>2064</td><td>Amount: 2</td></tr><tr><td>10 to 15 Series 2 Hole - Lite Transition Inside Corner Bracket</td><td>80/20</td><td>4509</td><td>Amount: 6</td></tr><tr><td>24&amp;rdquo;-long UV tube lights</td><td>ADJ Products LLC</td><td>T8-F20BLB24</td><td>Amount: 2&lt;br/&gt; 20W bulb &amp;ndash; 24&amp;rdquo; Wavelength: 365nm</td></tr><tr><td>Acrylic Mirror Sheet</td><td>Profesional Plastics</td><td></td><td>Amount: 1&lt;br/&gt; 82.5 cm x 26.5 cm</td></tr><tr><td>Acrylic Mirror Sheet</td><td>Profesional Plastics</td><td></td><td>Amount: 2&lt;br/&gt; 26.5 cm X 30.5 cm</td></tr><tr><td>Acrylic Mirror Sheet</td><td>Profesional Plastics</td><td></td><td>Amount: 2&lt;br/&gt; 82.5 cm x 30.5 cm</td></tr><tr><td>AR polycarbonate (UV resistance)</td><td>80/20</td><td>65-2641</td><td>Amount: 2&lt;br/&gt; 4.5mm Thick, Clear, 38.5 cm x 26.5 cm</td></tr><tr><td>AR polycarbonate (UV resistance)</td><td>80/20</td><td>65-2641</td><td>Amount: 4&lt;br/&gt; 4.5mm Thick, Clear, 38.5 cm x 21.5 cm</td></tr><tr><td>AR polycarbonate (UV resistance)</td><td>80/20</td><td>65-2641</td><td>Amount: 4&lt;br/&gt; 4.5mm Thick, Clear, 26.5 cm x 21.5 cm</td></tr><tr><td>AR polycarbonate (UV resistance)</td><td>80/20</td><td>65-2641</td><td>Amount: 4&lt;br/&gt; 4.5mm Thick, Clear 37.5 cm x 23.9 cm</td></tr><tr><td>AR polycarbonate (UV resistance)</td><td>80/20</td><td>65-2641</td><td>Amount: 4&lt;br/&gt; 4.5mm Thick, Clear , 24.4 cm x 23.9 cm</td></tr><tr><td>Chromatography paper (thin paper)</td><td>&amp;nbsp;Thermo Fisher Scientific</td><td>57144</td><td></td></tr><tr><td>Cosmos blotting paper (thick paper)</td><td>Blick Art Materials</td><td>10422-1005</td><td></td></tr><tr><td>Excel</td><td>Microsoft Corporation</td><td></td><td></td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software</td><td>Version 9.4.0</td><td>graphing and statistics software</td></tr><tr><td>ImageJ FIJI</td><td>NIH</td><td></td><td></td></tr><tr><td>Parafilm</td><td>Merck</td><td></td><td>transparent film</td></tr><tr><td>Quick Time Player 10.5 software&amp;nbsp;</td><td>Apple</td><td></td><td>multimedia player</td></tr><tr><td>Security spy</td><td>Ben software</td><td></td><td>video surveillance software system</td></tr><tr><td>Standard End Fastener, 1/4-20</td><td>80/20</td><td>3381</td><td>Amount: 80</td></tr><tr><td>UV transmitting acrylic</td><td>Spartech</td><td>Polycast Solacryl SUVT</td><td>Amount: 2&lt;br/&gt; 38.5 cm x 26.5 cm&amp;nbsp;</td></tr><tr><td>Water gel: HydroGel</td><td>ClearH2O&amp;nbsp;</td><td>&amp;nbsp;70-01-5022</td><td>(https://www.clearh2o.com/product/hydrogel/)</td></tr><tr><td>Webcam</td><td>Logitech</td><td>C930e</td><td>Amount: 4</td></tr></tbody></table>

real-time void spot assay

Normal voiding behavior is the result of the coordinated function of the bladder, the urethra, and the urethral sphincters under the proper control of the nervous system. To study voluntary voiding behavior in mouse models, researchers have developed the void spot assay (VSA), a method that measures the number and area of urine spots deposited on a filter paper lining the floor of an animal&#8217;s cage. Although technically simple and inexpensive, this assay has limitations when used as an end-point assay, including a lack of temporal resolution of voiding events and difficulties quantifying overlapping urine spots. To overcome these limitations, we developed a video-monitored VSA, which we call real-time VSA (RT-VSA), and which allows us to determine voiding frequency, assess voided volume and voiding patterns, and make measurements over 6 h time windows during both the dark and light phases of the day. The method described in this report can be applied to a wide variety of mouse-based studies that explore the physiological and neurobehavioral aspects of voluntary micturition in health and disease states.

Watch this Scientific Journal Video about Real-Time Void Spot Assay at JoVE.com

Real-Time Void Spot Assay

This article describes a new method to study mouse voiding behavior by incorporating video monitoring in the conventional void spot assay. This approach provides temporal, spatial, and volumetric information on the voiding events and details of mouse behavior during the light and dark phases of the day.

Normal voiding behavior is the result of the coordinated function of the bladder, the urethra, and the urethral sphincters under the proper control of the ...

Nanyang Technological University

Research

JoVE Journal

Behavior

1.8K Views.  University of Pittsburgh. This article describes a new method to study mouse voiding behavior by incorporating video monitoring in the conventional void spot assay. This approach provides temporal, spatial, and volumetric information on the voiding events and details of mouse behavior during the light and dark phases of the day.

Video: Real-Time Void Spot Assay

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

Environment

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

Cancer Research

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Biochemistry

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

Bioengineering

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of &#947;-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.

A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile

Here, we describe a method for purifying histidine-tagged pyrophosphokinase enzymes and utilizing thin layer chromatography of radiolabelled substrates and products to assay for the enzymatic activity in vitro. The enzyme activity assay is broadly applicable to any kinase, nucleotide cyclase, or phosphor-transfer reaction whose mechanism includes nucleotide triphosphate hydrolysis.

Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to ...

Immunology and Infection

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Biology

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to ...

Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay

Source: Tzeng, S. J. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54582">The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of Humoral Immunity.</a> J. Vis. Exp. (2016)
This video demonstrates the use of the ELISpot assay to assess the function of antibody-secreting B cells, ASCs. The antibodies secreted by the ASCs interact with the membrane-bound polyclonal antibody fragments, which are detected by the enzyme-conjugated IgG or IgM antibodies. The colored spot produced in each well indicates the presence of the specific antibody&#39;s isotype, which correlates with the B cells&#39; secretory activity.

Source: Tzeng, S. J. The Isolation, Differentiation, and Quantification of Human Antibody-secreting B Cells from Blood: ELISpot as a Functional Readout of ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Assays and Functional Profiling

Differential Radial Capillary Action of Ligand Assay: A High-Throughput Technique to Identify Bacterial Nucleotide Second Messenger-Binding Intracellular Proteins

Source: Schicketanz, M. L., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62331">Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA).</a> J. Vis. Exp. (2021)
This video demonstrates the differential radial capillary action of ligand assay &#8212; a technique for systematic high-throughput screening of intracellular nucleotide second messenger-binding proteins. This technique allows for direct binding of the messengers to the overexpressed target protein in the cell lysate &#8212; bypassing the need for protein purification.

Real-Time Void Spot Assay

Summary

Explore More Videos

Chapters in this video

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay

Differential Radial Capillary Action of Ligand Assay: A High-Throughput Technique to Identify Bacterial Nucleotide Second Messenger-Binding Intracellular Proteins