This work was supported by a grant from the Simons Foundation (SFARI #89638 to RB).

The following protocol adheres to the guidelines of the University of Washington institutional review board.
1. Electrophysiological Assessment
<ol>
	<li>Preparation of Session
	<ol>
		<li>Room preparation: place the manipulandum (see Figure 1), a wooden block with a sensor attached, which sends a time-stamped marker to acquisition software when it is grasped, on the table within grasping reach of the participant.&#160;Activate EEG acquisition software and begin &#8220;new session&#8221; (Figure S1).</li>
		<li>Net preparation: warm solution of distilled water (1 L), potassium chloride (1 tablespoon), and baby shampoo (1 teaspoon) to 104 &#176;F.&#160;Soak 128-electrode dense-array EEG system in the warmed saline solution.</li>
		<li>Participant preparation: ensure that the participant is seated comfortably approximately 75 cm from the stimulus presentation monitor and fully in view of the video camera.&#160;Find and mark the vertex on the participant&#8217;s head with a skin marker.&#160;Measure the vertex by finding the intersection of the midpoint between the nasion and the inion and the midpoint between preauriculars.</li>
		<li>Net application: Position the EEG cap on the participant&#8217;s head such that the vertex electrode is placed directly over the vertex mark.&#160;Check impedances and ensure that impedances are below the threshold appropriate for the EEG system in use (Figure S2).</li>
		<li>Begin video taping session.</li>
	</ol>
	</li>
	<li>Recording setup: Reference signal to the vertex electrode.&#160;Analog filter between 0.1 and 100 Hz, amplify the signal, and digitize at 500 samples/sec.</li>
	<li>Stimulus presentation: present participant with 3 conditions: observe, execute and rest, adapted from the paradigm developed by Muthukumaraswamy and colleagues5.
	<ol>
		<li>Observe condition: Instruct participant to sit quietly and watch a video of a person grasping the manipulandum.&#160;Each trial should last 6 sec. Time the prerecorded video for the observe trials precisely to ensure that the observed grasp occurs at exactly 3 sec. Monitor participant&#8217;s visual attention during the task, and mark trials during which they do not attend to the screen to be discarded during post-processing.</li>
		<li>Execute condition: Instruct participant to sit quietly with right hand resting just below the manipulandum and, upon hearing a prerecorded auditory cue, to imitate the manipulandum grab from the observe condition video clip. Each trial should last 6 sec. Ensure that the auditory cue is presented at exactly 3 sec by prerecording an auditory track that maintains a consistently timed execute cue and inter-trial interval.&#160;Utilize a sensor on the manipulandum to precisely record the time that the participant&#8217;s grasp occurs (Figure S3).</li>
		<li>Rest condition: Instruct participant to sit quietly with eyes open and passively observe a small crosshair on the stimulus monitor. Record continuous EEG during the rest condition for 3 min.</li>
		<li>For both observe and execute conditions, present randomized blocks of ten trials, for a total of forty trials per condition. Ensure that the image of the manipulandum remains on screen throughout the observe and execute blocks, including between trials. Administer the rest condition at the completion of the observe and execute conditions.</li>
	</ol>
	</li>
	<li>Data Processing
	<ol>
		<li>Following data collection, recheck impedances. Note any changes to impedance levels. End acquisition software recording.</li>
		<li>Post-processing: Rereference EEG signal to the average. Segment continuous EEG data into forty 6-sec trials for each condition (Figure S4).</li>
		<li>Conduct automated artifact detection. Use automated algorithms to inspect segments for movement artifacts by identifying fast average amplitudes exceeding 200 &#181;V, differential average amplitudes exceeding 100 &#181;V, and zero variance across a given trial (Figure S5).</li>
		<li>Conduct manual artifact detection by visually inspecting data and confirming with video review of the session to remove all trials in the observation condition contaminated with any movement artifact and all trials in the execution condition contaminated with any movement artifact unrelated to the grasp gesture. Exclude trials with significant artifact from analysis. Discard any trials that were flagged during acquisition as not attended. Examine and note rate of trial rejection for each diagnostic group under analysis.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Per Muthukumaraswamy et al.5, segment cleaned trials into 2 sec epochs consisting of 1 sec of data before the grasp and 1 sec after for both the observe (as marked by the photocell) and execute (as marked by the manipulandum sensor) conditions. Segment cleaned 2 sec epochs from the rest condition.&#160;</li>
		<li>Fast Fourier transform (FFT) each segment. Select a cluster of eight electrodes on each hemisphere surrounding the standard C3 and C4 positions for statistical analyses (following Muthukumaraswamy&#160;et al.5 and Bernier&#160;et al.3) (Figure 2). For each condition, average the power across the included trials to calculate power spectra.</li>
		<li>Calculate mu attenuation by examining the average power during either the execution or observation of a motor action, relative to the average power during the resting condition, across the 8-13 Hz range. Use the log of this ratio to determine degree of attenuation. Note: a negative value represents attenuation during execution or observation, while a positive value represents augmentation. This methodology takes into account variability across individuals, and the non-normality of values expressed in ratio form. 
		Note: This protocol was developed using a 128-electrode dense-array EEG system with Net Station software version 4.1. While the basic steps are similar across EEG systems, acquisition and analysis protocols may vary.</li>
	</ol>
	</li>
</ol>
2. Sample Characterization
<ol>
	<li>Identify potential patient population for participation in paradigm through research registries, previous participant listings, or referrals from area clinics and clinicians.</li>
	<li>Screen potential participants for likelihood of meeting diagnostic criteria for clinical construct (e.g. Autism Spectrum Disorder) and to identify any exclusionary criteria, such as presence of head injury, tumor, seizure history, or use of anti-convulsant or barbiturate medication which may distort the electrophysiological signal.</li>
	<li>Confirm diagnostic status of patient population through the use of gold standard diagnostic instruments (e.g. Autism Diagnostic Interview-Revised (ADI-R11) and the Autism Diagnostic Observation Schedule-Generic (ADOS-G,12) administered by expert clinician following Diagnostic and Statistical Manual &#8211; 5th Edition (DSM-5) criteria13.</li>
	<li>Identify control sample matched on relevant variables of interest, such as age, gender, cognitive ability, etc.</li>
</ol>

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The authors declare no competing financial interests.

The successful acquisition, processing, and analysis of electrophysiological data related to the mu rhythm and the application to clinical populations requires 1) the application of EEG methodological tools, 2) careful artifact detection and data reduction, 3) accurate identification of the mu rhythm, and 4) accurate characterization of the clinical population and identification of appropriate control groups.

Appropriate EEG methodology requires properly functionin...

Electroencephalography (EEG) is an effective, efficient, and noninvasive method of assessing and recording brain activity. As neurons fire in the brain, the resulting voltage can be amplified, recorded, and graphically represented. The temporal resolution of EEG allows for the analysis of even brief changes in the oscillation patterns of the brain, as well as the analysis of the brain&#8217;s response to specific stimuli.

Despite being the oldest brain imaging technique, dating back to the late 19th century, EEG still has wide-ranging applicability. While functional magnetic resonance imaging (fMRI) has excellent spatial resolution, it has relatively poor temporal resolution. This represents a major limitation of fMRI assessment given the incredible speed at which processes occur in the brain. EEG has the ability to assess electrical brain activity at the millisecond level, providing potential insight into the phases of the brain&#8217;s processing.

Evolving technologies have also expanded the applicability of EEG. An increase in the density of recording systems has allowed for the development of source localization techniques, mitigating some of EEG&#8217;s limitations regarding spatial resolution. Additionally, modern systems have reduced the individual participant set-up time significantly, allowing for the assessment of previously unavailable populations, such as infant and clinical samples1-3,28-30.

Given the excellent temporal resolution, EEG can be used to examine the neural response related to specific behaviors, states, or external stimuli. An example of this utility is the assessment of the mirror neuron system (MNS) in humans. Mirror neurons were originally identified in monkeys using single neuron recording4, evidencing a group of neurons that responded to both the execution and observation of motor actions. This direct recording method of placing electrodes in the brain is rarely utilized in humans, and only in dire clinical cases. EEG has provided a method for assessing the MNS by monitoring the EEG mu rhythm.&#160;This oscillation pattern in the 8-12 Hz range has been shown to attenuate EEG power in response to the execution and observation of motor actions, similar to the activation pattern observed in monkeys5-7. Similarly, stimulation of putative MNS brain regions through Transcranial Magnetic Stimulation (e.g.&#160;the inferior frontal gyrus) abolishes EEG mu rhythm8 and EEG mu rhythm suppression correlates with BOLD signals from fMRI in putative mirror neuron regions within subjects9, providing additional support that this rhythm indexes, at least in part, MNS activity. Assessment of the EEG mu rhythm has allowed for a noninvasive assessment of mirror neuron activity in humans.

EEG provides a unique methodology for examining brain activity and when combined with behaviorally based assays, it can be a powerful tool for elucidating aspects of social cognition, such as imitation, in clinical populations. Further, the applicability of EEG for use with populations with cognitive or language impairments allows for insight into abilities of individuals for whom other imaging techniques or behavioral paradigms may be less successfully utilize. The described protocol provides an avenue to examine social cognitive functions theoretically linked to the mirror neuron system in individuals with typical and atypical development, such as Autism Spectrum Disorder.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Geodesic EEG System</td>
			<td style="width:71px;">EGI</td>
			<td style="width:119px;">N/A</td>
			<td style="width:539px;">Any EEG system, not only EGI based systems, is applicable for the described study</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">MATLAB software</td>
			<td style="width:71px;">MATLAB</td>
			<td style="width:119px;">N/A</td>
			<td>Any mathematical, statistical software that can work with matrices is applicable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Netstation software</td>
			<td style="width:71px;">EGI</td>
			<td style="width:119px;">N/A</td>
			<td>Any EEG acquisition software is applicable for the described study</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Manipulandum</td>
			<td style="width:71px;">custom</td>
			<td style="width:119px;">N/A</td>
			<td>Any object that is co-registered with data acquisition software to signal a successful grasp</td>
		</tr>
	</tbody>
</table>

eeg mu rhythm in typical and atypical development

Electroencephalography (EEG) is an effective, efficient, and noninvasive method of assessing and recording brain activity. Given the excellent temporal resolution, EEG can be used to examine the neural response related to specific behaviors, states, or external stimuli. An example of this utility is the assessment of the mirror neuron system (MNS) in humans through the examination of the EEG mu rhythm. The EEG mu rhythm, oscillatory activity in the 8-12 Hz frequency range recorded from centrally located electrodes, is suppressed when an individual executes, or simply observes, goal directed actions. As such, it has been proposed to reflect activity of the MNS. It has been theorized that dysfunction in the mirror neuron system (MNS) plays a contributing role in the social deficits of autism spectrum disorder (ASD). The MNS can then be noninvasively examined in clinical populations by using EEG mu rhythm attenuation as an index for its activity. The described protocol provides an avenue to examine social cognitive functions theoretically linked to the MNS in individuals with typical and atypical development, such as ASD.&#160;

Typical adults, children and infants have consistently demonstrated mu rhythm during both the execution and observation of actions across a variety of paradigms and stimuli5,14-30. Attenuation in this frequency band is consistently localized across central electrodes (Figure 3) indicating that this is not reduction of alpha power which is recorded at other scalp regions. Similarly, attenuation in this frequency during the observation of movement is limited to the observat...

Watch this Scientific Journal Video about EEG Mu Rhythm in Typical and Atypical Development at JoVE.com

EEG Mu Rhythm in Typical and Atypical Development

Assessment of the EEG mu rhythm provides a unique methodology for examining brain activity and when combined with behaviorally based assays, can be a powerful tool for elucidating aspects of social cognition, such as imitation, in clinical populations.

Electroencephalography (EEG) is an effective, efficient, and noninvasive method of assessing and recording brain activity. Given the excellent temporal ...

eeg-mu-rhythm-in-typical-and-atypical-development

Research

JoVE Journal

Medicine

25.3K Views.  University of Washington. Assessment of the EEG mu rhythm provides a unique methodology for examining brain activity and when combined with behaviorally based assays, can be a powerful tool for elucidating aspects of social cognition, such as imitation, in clinical populations.

Video: EEG Mu Rhythm in Typical and Atypical Development

Direct Pressure Monitoring Accurately Predicts Pulmonary Vein Occlusion During Cryoballoon Ablation

Cryoballoon ablation (CBA) is an established therapy for atrial fibrillation (AF). Pulmonary vein (PV) occlusion is essential for achieving antral contact and PV isolation and is typically assessed by contrast injection. We present a novel method of direct pressure monitoring for assessment of PV occlusion.
Transcatheter pressure is monitored during balloon advancement to the PV antrum. Pressure is recorded via a single pressure transducer connected to the inner lumen of the cryoballoon. Pressure curve characteristics are used to assess occlusion in conjunction with fluoroscopic or intracardiac echocardiography (ICE) guidance. PV occlusion is confirmed when loss of typical left atrial (LA) pressure waveform is observed with recordings of PA pressure characteristics (no A wave and rapid V wave upstroke). Complete pulmonary vein occlusion as assessed with this technique has been confirmed with concurrent contrast utilization during the initial testing of the technique and has been shown to be highly accurate and readily reproducible.
We evaluated the efficacy of this novel technique in 35 patients. A total of 128 veins were assessed for occlusion with the cryoballoon utilizing the pressure monitoring technique; occlusive pressure was demonstrated in 113 veins with resultant successful pulmonary vein isolation in 111 veins (98.2%). Occlusion was confirmed with subsequent contrast injection during the initial ten procedures, after which contrast utilization was rapidly reduced or eliminated given the highly accurate identification of occlusive pressure waveform with limited initial training.
Verification of PV occlusive pressure during CBA is a novel approach to assessing effective PV occlusion and it accurately predicts electrical isolation. Utilization of this method results in significant decrease in fluoroscopy time and volume of contrast.

Effective pulmonary vein isolation utilizing a cryoballoon depends on complete pulmonary vein occlusion. The point of occlusion can be effectively predicted by direct analysis of pulmonary vein pressure waveform analysis during balloon inflation using a simple and reproducible technique.

Cryoballoon  ablation (CBA) is an established therapy for atrial fibrillation (AF).  Pulmonary vein (PV) occlusion is essential for achieving antral ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual&#39;s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized&#160;if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination&#160;of reporting animals as scrapie suspects. It&#160;could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.

A short clinical examination protocol is presented to aid in the detection of classical and atypical scrapie in sheep and goats.

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with focal epilepsy. This methodology relies on the assumption that seizures originate from the same regions of the brain from which interictal epileptiform discharges (IEDs) emerge. Preclinical models are very useful to find correlates between IED locations and the actual regions underlying seizure initiation in focal epilepsy. Rats have been commonly used in preclinical studies of epilepsy1; hence, there exist a large variety of models for focal epilepsy in this particular species. However, it is challenging to record multichannel EEG and to perform brain source imaging in such a small animal. To overcome this issue, we combine a patented-technology to obtain 32-channel EEG recordings from rodents2 and an MRI probabilistic atlas for brain anatomical structures in Wistar rats to perform brain source imaging. In this video, we introduce the procedures to acquire multichannel EEG from Wistar rats with focal cortical dysplasia, and describe the steps both to define the volume conductor model from the MRI atlas and to uniquely determine the IEDs. Finally, we validate the whole methodology by obtaining brain source images of IEDs and compare them with those obtained at different time frames during the seizure onset.

This video introduces the preparation, recording, and source analysis procedures of high-resolution EEG on sedated rats with a particular preclinical model of focal epilepsy under noninvasive conditions.

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with ...

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody (mAb) by Neutralizing TNF Using an In Vitro Bioanalytical Method

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an anti-TNF&#945; mAb. In addition, this protocol can be used to evaluate other anti-TNF&#945; molecules, such as fusion proteins. The cellular model employed here is sensitive to TNF&#945;-mediated apoptosis when an additional stress factor is induced in cell culture conditions (e.g., serum deprivation). This procedure exemplifies how to execute this analytical assay, highlighting the key operations relating to the sample preparation, cell dilution, apoptosis induction, and spectrophotometric measurements that are critical to ensure successful results. This protocol reveals the best-performance conditions relating to apoptosis induction and efficient signal recording, leading to low uncertainty values.

Determination of the Relative Potency of an Anti-TNF Monoclonal Antibody mAb by Neutralizing TNF Using an In Vitro Bioanalytical Method

A protocol for the determination of the relative anti-apoptotic activity of an anti-TNF&#945; mAb using a neutralization mechanism with WEHI 164 cells is presented here. This protocol is useful for comparing the neutralization strength of different molecules with the same biological functionality.

This protocol shows the measurement of the apoptotic activity neutralization of TNF&#945; in a mouse fibroblast cell model (WEHI 164) using an ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&amp;#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status epilepticus (SE) is frequently selected to recapitulate spontaneous recurrent seizures (SRS). Here we present a protocol of SE induction by intraperitoneal (i.p.) injection of pilocarpine and monitoring of chronic recurring seizures in live animals using a wireless telemetry video and electroencephalogram (EEG) system. We demonstrated notable behavioral changes that need attention after pilocarpine injection and their correlation with hippocampal neuronal loss at 7 days and 6 weeks post-pilocarpine. We also describe the experimental procedures of electrode implantation for video and EEG recording, and analysis of the frequency and duration of chronic recurrent seizures. Finally, we discuss the possible reasons why the expected results are not achieved in each case. This provides a basic overview of modeling chronic epilepsy in mice and guidelines for troubleshooting. We believe this protocol can serve as a baseline for suitable models of chronic epilepsy and epileptogenesis.

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...