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).

Urothelial&#160;Piezo1/2 double knockout mice (Pz1/2-KO, genotype: Piezo1fl/fl;Piezo2fl/fl;Upk2CRE+/-) and controls (Pz1/2-C, genotype: Piezo1fl/fl; Piezo2fl/fl; Upk2CRE-/-) were generated in-house from parental strains obtained from the Jax laboratories (Piezo1fl/fl strain # 029213; Piezo2fl/fl strain # 027720; Upk2CRE+/- strain # 029281). Both female (1.5&#8211;3 month...

<ol>
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Renal Physiology. 322 (1), 1 (2022).</li><li>Montalbetti, N., 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=Increased+urothelial+paracellular+transport+promotes+cystitis.">Increased urothelial paracellular transport promotes cystitis.</a> American Journal of Physiology. Renal Physiology. 309 (12), 1070 (2015).</li><li>Montalbetti, N., 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=Urothelial+tight+junction+barrier+dysfunction+sensitizes+bladder+afferents.">Urothelial tight junction barrier dysfunction sensitizes bladder afferents.</a> eNeuro. 4 (3), (2017).</li><li>Montalbetti, N., Stocker, S. D., Apodaca, G., Bastacky, S. I., Carattino, M. 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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 t...

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;

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		<tr>
			<td>1.00&#8221; X 1.00&#8221; T-Slotted Profile - Four Open T-Slots &#8211;&#160; cut to 10 inches</td>
			<td>80/20</td>
			<td>1010</td>
			<td>Amount: 20</td>
		</tr>
		<tr>
			<td>1.00&#8221; X 1.00&#8221; T-Slotted Profile - Four Open T-Slots &#8211;&#160; cut to 12 inches</td>
			<td>80/20</td>
			<td>1010</td>
			<td>Amount: 6</td>
		</tr>
		<tr>
			<td>1.00&#8221; X 1.00&#8221; T-Slotted Profile - Four Open T-Slots &#8211;&#160; cut to 40 inches</td>
			<td>80/20</td>
			<td>1010</td>
			<td>Amount: 4</td>
		</tr>
		<tr>
			<td>1.00&#8221; X 1.00&#8221; T-Slotted Profile - Four Open T-Slots &#8211; cut to 14.75 inches</td>
			<td>80/20</td>
			<td>1010</td>
			<td>Amount: 12</td>
		</tr>
		<tr>
			<td>1.00&#8221; X 1.00&#8221; T-Slotted Profile - Four Open T-Slots &#8211; 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 &#38; 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 - &#34;L&#34; Flat Plate</td>
			<td>80/20</td>
			<td>4081</td>
			<td>Amount: 8</td>
		</tr>
		<tr>
			<td>10 Series 5 Hole - &#34;T&#34; 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&#8221;-long UV tube lights</td>
			<td>ADJ Products LLC</td>
			<td>T8-F20BLB24</td>
			<td>Amount: 2 
			20W bulb &#8211; 24&#8221; Wavelength: 365nm</td>
		</tr>
		<tr>
			<td>Acrylic Mirror Sheet</td>
			<td>Profesional Plastics</td>
			<td></td>
			<td>Amount: 1 
			82.5 cm x 26.5 cm</td>
		</tr>
		<tr>
			<td>Acrylic Mirror Sheet</td>
			<td>Profesional Plastics</td>
			<td></td>
			<td>Amount: 2 
			26.5 cm X 30.5 cm</td>
		</tr>
		<tr>
			<td>Acrylic Mirror Sheet</td>
			<td>Profesional Plastics</td>
			<td></td>
			<td>Amount: 2 
			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 
			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 
			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 
			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 
			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 
			4.5mm Thick, Clear , 24.4 cm x 23.9 cm</td>
		</tr>
		<tr>
			<td>Chromatography paper (thin paper)</td>
			<td>&#160;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&#160;</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 
			38.5 cm x 26.5 cm&#160;</td>
		</tr>
		<tr>
			<td>Water gel: HydroGel</td>
			<td>ClearH2O&#160;</td>
			<td>&#160;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.

Voiding behavior of urothelial Piezo1/2 knockout mice
During the storage phase of the micturition cycle, the urothelium is hypothesized to sense the tension exerted by the urine accumulated in the bladder and to transduce this mechanical stimulus into cellular responses such as serosal ATP release1,3. We have previously shown that mechanically activated PIEZO1 and PIEZO2 channels are expressed in the mouse uro...

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 ...

The method described in this report can be used to understand physiological and neuro-behavioral aspects of voluntary micturition in health and disease states. This technique allows us to monitor mouse volume behavior in the light and dark phases of the day, and provides temporal, spatial, and volumetric information on voiding events. To prepare a real-time void spot assay or RT-VSA recording chamber, place a thin or thick filter paper, depending on the time of day, at the bottom of the RT-VSA recording cage.On top of the filter paper, place a plastic igloo for sleeping space;a sterile 1.5 ml microcentrifuge tube for enrichment purposes;and a 60 mm x 15 mm plastic dish containing two or three pieces of mouse dry chow;and 14 to 16 grams of water in the form of a gel pack. Once the recording chamber is ready, gently placed the mouse on the filter paper. Ensure that the transfer of the mouse from the housing cage to the recording cage occurs with minimum stress.Once the mouse is inside the recording cage, place the lid and cover the top of the lid with an absorbent bench blue pad to minimize direct ambient light reflections on the plexiglass lid surface. Turn on the ultraviolet lights in the lower chamber. To record RT-VSA video from the top and bottom cameras, use video surveillance recording software configured to record simultaneously from multiple webcams or networked cameras.Initiate recording by pressing Cmd R in the program window. Perform video recordings at one frame per second. Immediately after initiating the recordings, exit the room and close the door gently.Ensure that the room remains quiet for the entire experiment. Stop the recordings by pressing Cmd T.Turn off the ultraviolet lights. After stopping the recording, the software automatically generates a movie file in m4v format for each camera and saves it under the camera's name in a previously selected destination folder.Verify that within each camera's folder the experiments are organized into folders by date. In each date or experiment folder, verify that there is one m4v file and all of the individual jpeg files that correspond to each movie frame. Create a folder on the desktop with the name and date of the experiment and transfer the m4v files into this folder.Copy the movie folder into a flash drive for analysis on an external computer. Open a movie file collected by the bottom camera during the light phase or the upper camera during the dark phase for analysis. After assessing the quality of the movie, analyze experiments by moving to the correct time window using the fast forward command or the time bar slider.Voiding activity during the light phase is recorded between 11:00 AM and 5:00 PM, and during the dark phase, between midnight and 6:00 AM.Play the movie in fast forward mode by clicking on the fast forward icon or manually scroll through the movie, looking for evidence that the mouse is voiding. Look for the sudden appearance of bright urine spots on the filter paper or the behavioral changes, including movement to the corners of the cage and a brief period of inactivity when the mouse is voiding. Register the time at which each void occurs.As a convention, the time of the void is recorded at the first sign the urine is detected. To take measurements of the void, first use the scroll bar to move forward or backward in time, looking for the point when maximal diffusion of the urine spot has occurred. Pause the movie at this point and place the computer mouse arrow at the spot under analysis to mark the spot of interest in the screenshot.Name the screenshot file using correlative numbers to account for the order of appearance in the movie. Once all the void spots have been analyzed, measure the total area of the filter paper by capturing a screenshot and delineating the border of the filter paper. Calculate the percentage area of each of the urine spots.Transform the values of the percentage area into the volume of urine for each void spot using the calibration curves and the interpolate function in the graphing software. The voiding behavior of female and male Piezo1, 2, knockout and control mice were recorded during the dark and light phase of the day. During their inactive light phase, no significant differences were observed in any parameters analyzed for female or male knockout mice compared to the control.However, in the active dark phase, female and male knockout mice showed altered voiding phenotype, characterized by a significant increase in the number and total volume of small void spots. There was no significant differences in primary voids spots or PVS number, average volume per PVS, or total PVS volume in female or male knockout mice compared to the control mice. The voiding behavior of female mice was tested under basal conditions and following cyclophosphamide treatment.Compared to the basal conditions, the amount of urine release per void is smaller after cyclophosphamide treatment and the micturition events are much more frequent. One key aspect for assay reproducibility is to manipulate the animals as gently as possible and keep them under minimum stress during the whole test period. As this is an noninvasive assay, additional procedures can be performed afterward including cytometry and electromyography to assess bladder and external urethra sphincter functions respectively.Implementing this technique will allow researchers to define the contribution of specific pathways to voiding behavior in health and disease states.

Research

JoVE Journal

Behavior

1.7K ビュー.  University of Pittsburgh. このレポートに記載されている方法は、健康状態および疾患状態における自発的排尿の生理学的および神経行動的側面を理解するために使用できます。この手法により、その日の明るい段階と暗い段階でマウスのボリュームの動作を監視でき、排尿イベントに関する時間的、空間的、および体積情報が提供されます。リアルタイムボイドスポットアッセイまたはRT-VSA記?...

ビデオ: 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...

real-time polymorphonuclear neutrophils (pmn) chemotaxis assay

Real-Time Polymorphonuclear Neutrophils (PMN) Chemotaxis Assay  

quantitative real-time pcr using the thermo scientific solaris qpcr assay

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the...

qpcrtag analysis - a high throughput, real time pcr assay for sc2.0 genotyping

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

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...

light spot-based assay for analysis of drosophila larval phototaxis

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

This protocol introduces a light-spot assay to investigate Drosophila larval phototactic behavior. In this assay, a light spot is generated as light stimulation, and the process of larval light avoidance is recorded by an infrared light-based imaging...

Light Spot-Based Assay for Analysis of Drosophila Larval Phototaxis

live-cell measurement of odorant receptor activation using a real-time camp assay

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP...

real-time in vitro migration assay for primary murine cd8+ t cells

Author Spotlight: Understanding Disease Mechanisms Through Real-Time Analysis of T-Cell Migration

This protocol providesa method of primary murine T cell isolation and time-lapse microscopy of T cell migration under specific environmental conditions with quantitative...

Real-Time In Vitro Migration Assay for Primary Murine CD8+ T Cells

spot the correct tissue every time in multi-tissue blocks

SpOT the Correct Tissue Every Time in Multi-tissue Blocks

The purpose of the Specimen Orientation Tag (SpOT) is to function as an orientation tool to aid in individual tissue identification in multi-tissue paraffin blocks. These protocols demonstrate how it is constructed easily from common, low-cost histology materials and serves as a reliable visual marker in paraffin blocks and...

light spot assay: a method to investigate drosophila phototactic behavior

Drosophila larvae are light-sensitive and respond to changes in surrounding light. This video describes an assay that tests their ability to avoid light, called the light spot assay. The featured protocol demonstrates how to set up the assay with a blue light LED and shows how to record the animal&#39;s behavior in response to the light...

Light Spot Assay: A Method to Investigate Drosophila Phototactic Behavior

real-time quaking-induced conversion assay for detection of cwd prions in fecal material

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC)...

an assay for real-time monitoring of extracellular ph in dental biofilms

This video demonstrates a real-time assay for monitoring extracellular pH in dental biofilms using a pH-sensitive ratiometric probe. When an in situ-grown bacterial biofilm is exposed to glucose, a reduction in extracellular pH near the bacterial biomass protonates the probe, leading to its increased concentration inside the bacteria. The disparity in emission wavelength between the protonated and unprotonated probe is utilized to calculate the pH across the extracellular matrix of the biofilm...

An Assay for Real-Time Monitoring of Extracellular pH in Dental Biofilms

Add 5 microliters of the ratiometric dye per well. Place the 96-well plate on the microscope stage. Then, turn on the microscope, and the 543 laser line. Warm up the incubator to 37 degrees Celsius. Use the same microscope settings as for the calibration of the...

a spot plate assay for evaluating oxidative stress responses in bacterial strains

This video demonstrates a spot plate assay tailored to assess the oxidative stress responses of bacterial strains. Some strains produce pyomelanin, an antioxidant pigment. Conversely, strains treated with a test agent, leading to reduced pyomelanin production, exhibit increased susceptibility to oxidative stress, resulting in decreased cell viability compared to pyomelanin-producing...

A Spot Plate Assay for Evaluating Oxidative Stress Responses in Bacterial Strains

a real-time in vitro cytolysis assay to evaluate the potency of car t cells

This video demonstrates a method for monitoring CAR T cell activity against cancer cells through real-time electrical impedance measurements. Tracking changes in impedance assesses the effectiveness of CAR T cells in inducing cancer cell cytolysis, providing insights into their cytotoxic...

A Real-Time In Vitro Cytolysis Assay to Evaluate the Potency of CAR T Cells

a protocol for real-time 3d single particle tracking

A Protocol for Real-time 3D Single Particle Tracking

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count...

assay to measure nucleocytoplasmic transport in real time within motor neuron-like nsc-34 cells

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

This protocol describes a method to sensitively measure the nucleocytoplasmic transport rate within motor neuron-like NSC-34 cells by quantifying the real-time change in the nuclear import of a NLS-NES-GFP...

Detach the target cancer cells from the culture plate using trypsin. Wash the cells in 15 milliliters of fresh culture medium and centrifuge. Re-suspend the cancer cell pellet in 5 milliliters of fresh medium for counting and adjust the cell density to the appropriate target cell concentration. Add the cells to the appropriate number of wells, and leave the E-plate for 30 minutes at room temperature to allow the cells to settle evenly on the bottom of the...

a real-time potency assay for chimeric antigen receptor t cells targeting solid and hematological cancer cells

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination...

real-time detection of reactive oxygen species production in immune response in rice with a chemiluminescence assay

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay

Here, we describe a method for the real-time detection of apoplastic reactive oxygen species (ROS) production in rice tissues in pathogen-associated molecular pattern-triggered immune response. This method is simple, standardized, and generates highly reproducible results under controlled...

real-time fmri brain mapping in animals

Real-Time fMRI Brain Mapping in Animals

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal...

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for quantifying locomotor deficits in rodents is to measure minute disturbances of unconstrained gait overground (e.g., manual BBB score or automated CatWalk). However, cortical inputs are not required for the generation of basic locomotion produced by the spinal central pattern generator (CPG). Thus, unconstrained walking tasks test locomotor deficits due to motor cortical impairment only indirectly. In this study, we propose a novel, precise foot-placement locomotor task that evaluates cortical inputs to the spinal CPG. An instrumented peg-way was used to impose symmetrical and asymmetrical locomotor tasks mimicking lateralized movement deficits. We demonstrate that shifts from equidistant inter-stride lengths of 20% produce changes in the forelimb stance phase characteristics during locomotion with preferred stride length. Furthermore, we propose that the asymmetric walkway allows for measurements of behavioral outcomes produced by cortical control signals. These measures are relevant for the assessment of impairment after cortical damage.

Here, we present a protocol to quantify precise stepping in rodents. Cortical and the spinal central pattern generator signals are required for precise foot-placement during obstructed locomotion. We report here the novel constrained walking task that directly examines precise stepping behavior.

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the appearance and behavior of visual stimuli, compared to manipulating live animals. Here, we present the FishSim Animation Toolchain, a software framework developed to provide researchers with an easy-to-use method for implementing 3D computer animations in behavioral experiments with fish. The toolchain offers templates to create virtual 3D stimuli of five different fish species. Stimuli are customizable in both appearance and size, based on photographs taken of live fish. Multiple stimuli can be animated by recording swimming paths in a virtual environment using a video game controller. To increase standardization of the simulated behavior, the prerecorded swimming path may be replayed with different stimuli. Multiple animations can later be organized into playlists and presented on monitors during experiments with live fish.
In a case study with sailfin mollies (Poecilia latipinna), we provide a protocol on how to conduct a mate-choice copying experiment with FishSim. We utilized this method to create and animate virtual males and virtual model females, and then presented these to live focal females in a binary choice experiment. Our results demonstrate that computer animation may be used to simulate virtual fish in a mate-choice copying experiment to investigate the role of female gravid spots as an indication of quality for a model female in mate-choice copying.
Applying this method is not limited to mate-choice copying experiments but can be used in various experimental designs. Still, its usability depends on the visual capabilities of the study species and first needs validation. Overall, computer animations offer a high degree of control and standardization in experiments and bear the potential to &#39;reduce&#39; and &#39;replace&#39; live stimulus animals as well as to &#39;refine&#39; experimental procedures.

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish.

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the ...

Using a Real-Time Locating System to Measure Walking Activity Associated with Wandering Behaviors Among Institutionalized Older Adults

A real-time locating system (RTLS) can be used to track the walking activity of institutionalized older adults in long-term care who are at risk for wandering behaviors. The benefits of a RTLS are objective and continuous measurements of activity. Self-report methods of activity, especially wandering, by health care staff are vulnerable to floor effects and recall bias, and continuous clinical or research observation over the long-term can be time-consuming and expensive. Health care staff also fail to recognize the onset and/or duration of wandering behaviors, which are associated with a variety of adverse health outcomes in this population but amenable to intervention. RTLS technologies can measure the walking activity of institutionalized residents with cognitive impairment over time with a high degree of accuracy. This is particularly useful for the study of wandering, defined as walking for at least 60 seconds with few (if any) breaks in activity. Wandering is associated with disease progression, hospitalizations, falls and death. Previous work suggests older adults with poor balance ability and high sustained walking activity may be particularly susceptible to poor health outcomes. RTLS&#39;s are used to assess cognitive impairment and factors associated with gait and balance; however, supplemental paper and pencil gait/balance tools may be used to further refine risk profiles. This project discusses the use of a RTLS to measure walking activity and also gait quality and balance ability measures on this population.

This paper discusses the use of a continuous and objective real-time locating system to measure walking activity associated with wandering behaviors, focusing on older adults with cognitive impairment. Walking activity is measured by walking distance, sustained walking distance, and sustained gait speed. Also assessed are gait quality and balance ability.

A real-time locating system (RTLS) can be used to track the walking activity of institutionalized older adults in long-term care who are at risk for ...

Brain State-dependent Brain Stimulation with Real-time Electroencephalography-Triggered Transcranial Magnetic Stimulation

The effect of a stimulus to the brain depends not only on the parameters of the stimulus but also on the dynamics of brain activity at the time of the stimulation. The combination of electroencephalography (EEG) and transcranial magnetic stimulation (TMS) in a real-time brain state-dependent stimulation system allows the study of relations of dynamics of brain activity, cortical excitability, and plasticity induction. Here, we demonstrate a newly developed method to synchronize the timing of brain stimulation with the phase of ongoing EEG oscillations using a real-time data analysis system. This real-time EEG-triggered TMS of the human motor cortex, when TMS is synchronized with the surface EEG negative peak of the sensorimotor &#181;-alpha (8-14 Hz) rhythm, has shown differential corticospinal excitability and plasticity effects. The utilization of this method suggests that real-time information about the instantaneous brain state can be used for efficacious plasticity induction. Additionally, this approach enables personalized EEG-synchronized brain stimulation which may lead to the development of more effective therapeutic brain stimulation protocols.

This paper describes real-time electroencephalography-triggered transcranial magnetic stimulation to study and modulate human brain networks.

The effect of a stimulus to the brain depends not only on the parameters of the stimulus but also on the dynamics of brain activity at the time of the ...

A Cross-Disciplinary and Multi-Modal Experimental Design for Studying Near-Real-Time Authentic Examination Experiences

Over the past ten years, research into students&#39; emotions in educational environments has increased. Although researchers have called for more studies that rely on objective measures of emotional experience, limitations on utilizing multi-modal data sources exist. Studies of emotion and emotional regulation in classrooms traditionally rely on survey instruments, experience-sampling, artifacts, interviews, or observational procedures. These methods, while valuable, are mainly&#160;dependent on participant or observer subjectivity and is limited in its authentic measurement of students&#39; real-time performance to a classroom activity or task. The latter, in particular, poses a stumbling block to many scholars seeking to objectively measure emotions and other related measures in the classroom, in real-time.
The purpose of this work is to present a protocol to experimentally study students&#39; real-time responses to exam experiences during an authentic assessment situation. For this, a team of educational psychologists, engineers, and engineering education researchers designed an experimental protocol that retained the limits required for accurate physiological sensor measurement, best-practices of salivary collection, and an authentic testing environment. In particular, existing studies that rely on physiological sensors are conducted in experimental environments that are disconnected from educational settings (e.g., Trier Stress Test), detached in time (e.g., before or after a task), or introduce analysis error (e.g., use of sensors in environments where students are likely to move). This limits our understanding of students&#39; real-time responses to classroom activities and tasks. Furthermore, recent research has called for more considerations to be covered around issues of recruitment, replicability, validity, setups, data cleaning, preliminary analysis, and particular&#160;circumstances (e.g., adding a variable in the experimental design) in academic emotions research that relies on multi-modal approaches.

An experimental design was developed to investigate the real-time influences of an examination experience to assess the emotional realities students experience in higher education settings and tasks. This design is the result of a cross-disciplinary (e.g., educational psychology, biology, physiology, engineering) and multi-modal (e.g., salivary markers, surveys, electrodermal sensor) approach.

Over the past ten years, research into students&#39; emotions in educational environments has increased. Although researchers have called for more studies ...

Implementation of a Real-Time Psychosis Risk Detection and Alerting System Based on Electronic Health Records using CogStack

Recent studies have shown that an automated, lifespan-inclusive, transdiagnostic, and clinically based, individualized risk calculator provides a powerful system for supporting the early detection of individuals at-risk of psychosis at a large scale, by leveraging electronic health records (EHRs). This risk calculator has been externally validated twice and is undergoing feasibility testing for clinical implementation. Integration of this risk calculator in clinical routine should be facilitated by prospective feasibility studies, which are required to address pragmatic challenges, such as missing data, and the usability of this risk calculator in a real-world and routine clinical setting. Here, we present an approach for a prospective implementation of a real-time psychosis risk detection and alerting service in a real-world EHR system. This method leverages the CogStack platform, which is an open-source, lightweight, and distributed information retrieval and text extraction system. The CogStack platform incorporates a set of services that allow for full-text search of clinical data, lifespan-inclusive, real-time calculation of psychosis risk, early risk-alerting to clinicians, and the visual monitoring of patients over time. Our method includes: 1) ingestion and synchronization of data from multiple sources into the CogStack platform, 2) implementation of a risk calculator, whose algorithm was previously developed and validated, for timely computation of a patient&#39;s risk of psychosis, 3) creation of interactive visualizations and dashboards to monitor patients&#39; health status over time, and 4) building automated alerting systems to ensure that clinicians are notified of patients at-risk, so that appropriate actions can be pursued. This is the first ever study that has developed and implemented a similar detection and alerting system in clinical routine for early detection of psychosis.

We demonstrate how to deploy a real-time psychosis risk calculation and alerting system based on CogStack, an information retrieval and extraction platform for electronic health records.

Recent studies have shown that an automated, lifespan-inclusive, transdiagnostic, and clinically based, individualized risk calculator provides a powerful ...

Two Different Real-Time Place Preference Paradigms Using Optogenetics within the Ventral Tegmental Area of the Mouse

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with behavioral testing in validated paradigms allows the measurement of behavioral consequences upon stimulation of distinct neurons in real-time with high spatial and temporal selectivity, and thus the establishment of causal relationships between neuronal activation and behavior. Here, we describe a step-by-step protocol for a real-time place preference (RT-PP) paradigm, a modified version of the classical conditioned place preference (CPP) test. The RT-PP is performed in a three-compartment apparatus and can be utilized to answer if optogenetic stimulation of a specific neuronal population is rewarding or aversive. We also describe an alternative version of the RT-PP protocol, the so-called neutral compartment preference (NCP) protocol, which can be used to confirm aversion. The two approaches are based on extensions of classical methodology originating from behavioral pharmacology and recent implementation of optogenetics within the neuroscience field. Apart from measuring place preference in real time, these setups can also give information regarding conditioned behavior. We provide easy-to-follow step-by-step protocols alongside examples of our own data and discuss important aspects to consider when applying these types of experiments.

Here we present two easy-to-follow step-by-step protocols for place preference paradigms using optogenetics in mice. Using these two different setups, preference and avoidance behaviors can be solidly assessed within the same apparatus with high spatial and temporal selectivity, and in a straightforward manner.

Understanding how neuronal activation leads to specific behavioral output is fundamental for modern neuroscience. Combining optogenetics in rodents with ...

Real-Time Proxy-Control of Re-Parameterized Peripheral Signals using a Close-Loop Interface

The fields that develop methods for sensory substitution and sensory augmentation have aimed to control external goals using signals from the central nervous systems (CNS). Less frequent however, are protocols that update external signals self-generated by interactive bodies in motion. There is a paucity of methods that combine the body-heart-brain biorhythms of one moving agent to steer those of another moving agent during dyadic exchange. Part of the challenge to accomplish such a feat has been the complexity of the setup using multimodal bio-signals with different physical units, disparate time scales and variable sampling frequencies.
In recent years, the advent of wearable bio-sensors that can non-invasively harness multiple signals in tandem, has opened the possibility to re-parameterize and update the peripheral signals of interacting dyads, in addition to improving brain- and/or body-machine interfaces. Here we present a co-adaptive interface that updates efferent somatic-motor output (including kinematics and heart rate) using biosensors; parameterizes the stochastic bio-signals, sonifies this output, and feeds it back in re-parameterized form as visuo/audio-kinesthetic reafferent input. We illustrate the methods using two types of interactions, one involving two humans and another involving a human and its avatar interacting in near real time. We discuss the new methods in the context of possible new ways to measure the influences of external input on internal somatic-sensory-motor control.

We present protocols and methods of analyses to build co-adaptive interfaces that stream, parameterize, analyze, and modify human body and heart signals in close-loop. This setup interfaces signals derived from the peripheral and central nervous systems of the person with external sensory inputs to help track biophysical change.

The fields that develop methods for sensory substitution and sensory augmentation have aimed to control external goals using signals from the central ...

Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae

Gravity sensation is an important and relatively understudied process. Sensing gravity enables animals to navigate their surroundings and facilitates movement. Additionally, gravity sensation, which occurs in the mammalian inner ear, is closely related to hearing - thus, understanding this process has implications for auditory and vestibular research. Gravitaxis assays exist for some model organisms, including Drosophila. Single worms have previously been assayed for their orientation preference as they settle in solution. However, a reliable and robust assay for Caenorhabditis gravitaxis has not been described. The present protocol outlines a procedure for performing gravitaxis assays that can be used to test hundreds of Caenorhabditis dauers at a time. This large-scale, long-distance assay allows for detailed data collection, revealing phenotypes that may be missed on a standard plate-based assay. Dauer movement along the vertical axis is compared with horizontal controls to ensure that directional bias is due to gravity. Gravitactic preference can then be compared between strains or experimental conditions. This method can determine molecular, cellular, and environmental requirements for gravitaxis in worms.

Large-Scale Gravitaxis Assay of Caenorhabditis Dauer Larvae

The present protocol outlines methods for conducting a large-scale gravitaxis assay with Caenorhabditis dauer larvae. This protocol allows for better detection of gravitaxis behavior compared with a plate-based assay.

Gravity sensation is an important and relatively understudied process. Sensing gravity enables animals to navigate their surroundings and facilitates ...