The human intestinal enteroids were obtained from the lab of Dr. Michael Helmrath at Cincinnati Children&#39;s Hospital Medical Center (CCHMC). This work was supported by R01 DK11005 (CIH) and the University of Cincinnati Cancer Center Pilot Funding. We are grateful for imaging support from the University of Cincinnati Live Microscopy Core (NIH S10OD030402).

All experiments using human tissues for the generation of HIEs were approved by an IRB at CCHMC (IRB#2014-0427). See the Table of Materials for details related to all materials used in this protocol.
NOTE: To illustrate the procedure outlined in this protocol, we utilized Bmal1-luc human intestinal enteroids (HIEs). These enteroids underwent stable lentiviral transduction22 with the pABpuro-BluF reporter plasmid, which contains the Bmal1 promoter fused to luciferase, showing the activity of the Bmal1 promoter21.
1. Lentiviral transduction
<ol>
	<li>Carry out the lentiviral transduction22 under stem cell-enriched conditions for HIEs. Specifically, ensure the formation of spheroids in HIEs within 3-4 days after passage in the intestinal growth media, which creates stem cell-enriched conditions.</li>
	<li>After the lentiviral transduction22, carry out antibiotic selection with 2 &#181;g/mL puromycin, recover the puromycin-resistant HIEs, and passage them with relatively high density (i.e., 1 well into 2 wells) for 2-3x. If the growth rate of the HIEs is slow, add 10 &#181;M ROCK inhibitors (Y-27632) and 10 &#181;M GSK-3 inhibitor (CHIR-99021) to the intestinal organoid growth medium.</li>
</ol>
2. Preparation of organoids for bioluminescence assay
<ol>
	<li>Day 1: Passage HIEs to 35 mm round dishes.
	<ol>
		<li>Briefly, for expansion of HIEs, mix the HIEs with growth factor-reduced (GFR) 3D culture matrix and seed them onto 24-well plates with three droplets of 10 &#181;L of 3D culture matrix per well. Add 350 &#181;L of the HIE growth medium and change it every other day. 
		NOTE: The density of HIE in the 3D culture matrix is critical for the growth of enteroids. Approximately, the number of spheroids should exceed more than 50 in each droplet. If the number is low, the growth rate of the HIE is negatively affected.</li>
		<li>When HIEs grow sufficiently (approximately 7 days after the post-passage), collect HIEs from four wells into 1.5 mL microcentrifuge tubes.</li>
		<li>Wash the HIEs with the cold PBS using a benchtop mini centrifuge (2,000 &#215; g) for 40 s quick spin to remove debris and excessive 3D culture matrix by pipet.</li>
		<li>Add 0.5 mL of the cold PBS into the pellet and apply physical turbulence by syringing up to 10x with a 1 mL insulin syringe to dissociate the HIEs.</li>
		<li>Spin down the dissociated HIEs with a benchtop mini centrifuge as in step 2.1.3 and gently remove the supernatant by pipet. Then, place the microcentrifuge tube with the HIE pellet on ice to cool down for approximately 3 min.</li>
		<li>Add GFR 3D culture matrix to the microcentrifuge tube and mix it with the HIE pellet. 
		NOTE: Calculate the amount of the 3D culture matrix based on the number of 35 mm round dishes that will be used for the experiment. We generally seed 30 &#181;L of 3D culture matrix droplets per round dish.</li>
		<li>Seed the mixture of HIEs and the 3D culture matrix on the center of the 35 mm round dish and incubate the dish at 37 &#176;C for 20 min to solidify the 3D culture matrix. 
		NOTE: The density of seeding HIEs should be determined experimentally. Based on the intensity of bioluminescence, one can determine the number of HIEs during this process.</li>
		<li>Add 2 mL of prewarmed intestinal organoid growth medium to each dish and incubate the dishes in a CO2 incubator until the next step.</li>
	</ol>
	</li>
	<li>Day 3: Initiation of differentiation
	<ol>
		<li>To initiate differentiation of the HIEs, remove the growth medium and add 2 mL of prewarmed differentiation medium. 
		NOTE: See Table 1 for the recipes of the HIE differentiation media23.</li>
	</ol>
	</li>
	<li>Day 4: No additional process is required.</li>
	<li>Day 5: Replace the medium of the enteroids with 2 mL of prewarmed differentiation medium.</li>
	<li>Day 6: Clock synchronization and recording bioluminescence
	<ol>
		<li>To synchronize the molecular clock of enteroids, add 2 &#181;L of 100 &#181;M dexamethasone (Dex) to make the final concentration 100 nM in each dish and then incubate the dishes in a CO2 incubator (37 &#176;C, 5% CO2) for 1 h24.</li>
		<li>During the Dex resetting, prepare the incubating luminometer. Check the CO2 and temperature of the incubating luminometer and set to 5% and 37 &#176;C, respectively. 
		NOTE: It is suggested to wipe the inside of the incubating luminometer with 70% alcohol before and after every use of the machine to keep it sterile.</li>
		<li>After the clock synchronization, replenish the enteroids with 3 mL of prewarmed differentiation medium containing 200 &#181;M D-luciferin. 
		NOTE: As luciferin is light-sensitive, the luciferin-containing medium should be wrapped with foil and prewarmed in a 37 &#176;C heat bath before applying it to the dish.</li>
		<li>Place the dishes in an 8-well dish table of the incubating luminometer and cover the sample dish table with its lid. Place two humidifier chambers with wet tissue or sponge on the top of the sample dish table. This is one-time process. 
		NOTE: After the experiment is started, the lid of the incubating luminometer should be kept closed until the experiment is terminated. Make sure to wet the tissues/sponges enough.</li>
		<li>Close the lid of the machine and initiate the program to measure bioluminescence for the desired number of days and resolution.
		<ol>
			<li>On the software, select the condition settings by clicking the Condition tab and adjust the settings on this panel: number of dishes, measurement time, background processing, and filter type. 
			NOTE: For this demonstration, we selected all dishes from A to H; default measurement time, which is determined by the software based on the number of dishes; waiting time for background processing, and filter type F0. Since there are three filter options, three luminescent colors can be measured simultaneously.</li>
			<li>After entering all suitable settings based on the experiment, save the settings by clicking OK.</li>
			<li>To start running the experiment, click the Run tab | Start button (green triangle icon). Observe the data as raw, noise filtered, or detrended by making appropriate selections on the top of the screen of the software while running experiments. Click on Raw, Noise Filtered, or Detrended tabs to observe the selected data.</li>
			<li>Observe signals for up to 5 days. 
			NOTE: After 5 days of monitoring in an incubating luminometer, enteroids may begin to die due to media depletion. To prevent any possible circadian disruption during the experiment, we do not perform media change for the duration of the experiment.</li>
			<li>At the end of the experiment, stop the run by clicking the Stop button (blue square icon), go to the File tab, and click Save as. Save the data in the Kronos format; export the data to a spreadsheet in the File section by clicking the File option and then selecting Export as excel format under the File for further analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

Optimizing Human Intestinal Enteroids for Circadian Rhythm Studies

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The authors declare that they have no conflicts of interest.

Bioluminescence assay offers several advantages for the investigation of circadian rhythms, which requires data collection from long-term time course experiments. First, it enables researchers to monitor the gene expression or protein of interest as cells move and proliferate. Without making unnecessary adjustments or disrupting the cells&#39; functions, interested cellular events or gene expression can be recorded using bioluminescence readout, which gives reliable real-time data. Importantly, the bioluminescence assay ...

Circadian clock 
All organisms, from bacteria to mammals, have a complex and dynamic relationship with their environment. Within this relationship, adaptation to environmental changes is critical for the survival of organisms. Most organisms possess circadian rhythms that enable them to adapt and optimize their functions to diurnal cycles of approximately 24 h. The circadian clock is a hierarchical network of central and peripheral clocks that work in cooperation to maintain physiological homeostasis and keep organisms synchronized with daily changes1,2. In mammals, the central or master clock located in the suprachiasmatic nucleus (SCN) receives external cues, such as light, and transmits the information to the peripheral clocks via an advanced interplay of neural and humoral signaling pathways3. In addition to the central clock, peripheral tissues possess their own cell-autonomous circadian clock mechanism, maintained by a transcriptional-translational negative feedback loop (TTFL) regulating tissue-specific clock-controlled genes (CCGs)4,5. This molecular machinery produces approximately 24 h rhythmicity in cellular and physiological events, such as gene expressions, signaling pathways, immune responses, and digestion. The circadian clock is present in almost all mammalian cells, and it has been demonstrated that up to 50% of the genes&#39; expression patterns exhibit circadian rhythmicity6. Considering the abundance of CCGs, disruption of this clock mechanism may result in critical physiological problems. Hence, investigations into circadian rhythms are necessary to elucidate essential biological mechanisms and develop novel therapeutic strategies.
Luciferase reporter system 
In circadian studies, real-time monitoring is critical for a better understanding of cellular behaviors and responses because it allows tracking of temporal changes in gene expression and/or protein levels, providing insights into the molecular mechanisms regulated by the circadian clock. Furthermore, real-time monitoring enables researchers to study the effects of environmental changes on molecular mechanisms7,8. There are numerous techniques for real-time monitoring studies, including bioluminescence assay, which is widely used to track gene expression or protein levels over time. Bioluminescence assay is a method to detect biological processes using light production as a readout. In this assay, an oxidative enzyme that produces bioluminescence (e.g., luciferase) is either transiently or stably transfected into cells of interest, and the bioluminescence readout is measured in the presence of a substrate (e.g., luciferin) over time. For example, the luciferase enzyme produces bioluminescence by oxidizing the substrate luciferin in the presence of ATP9. Due to its short half-life, 3-4 h10, firefly luciferase is a powerful tool for circadian studies in terms of providing real-time dynamic monitoring with minimal background noise11,12,13. For the insertion of DNA with a luciferase-tagged promoter or open reading frame (ORF), the lentiviral gene delivery system is a reliable method that provides high transduction efficacy, stable integration, and low immunogenicity. Stable transduction of a bioluminescent reporter provides robust expression in dividing and non-dividing cells, generating consistent data for circadian studies14.
Organoid as a model 
Traditional immortalized two-dimensional cell lines have been instrumental in biological studies ranging from uncovering fundamental molecular mechanisms of circadian rhythms to drug screening. Despite the convenience of utilizing homogenized cell lines, they lack multicellular structures and intercellular interactions. In contrast, organoids are in vitro 3D multicellular &#34;organ-like&#34; structures that mimic organ structure in a dish by displaying similarity to in vivo tissue architecture and multicellularity, including stem, progenitor, and differentiated cell types15,16. Possessing self-organization, multicellularity, and functionality features makes organoids a remarkable in vitro model representing the cellular and physiological processes occurring in real tissues17. Different types of organoids can be derived from pluripotent stem cells via directed differentiation or adult stem cells harvested from various organs, including the small intestine, brain, liver, lung, and kidney18,19. Since organoid structures possess a real tissue-like architecture and function with multicellularity and dynamic cell-to-cell interaction, they are superior to homogenized cell lines for understanding the cellular events occurring in in vivo tissues. Organoids are also easily manipulated and can be grown under controlled conditions, making them useful for circadian studies20.
The main purpose of this work is to introduce a real-time monitoring method utilizing a bioluminescence assay specifically tailored for studying circadian rhythms in multicellular 3D organoids. Real-time monitoring of cellular events using a bioluminescence assay technique has been widely performed for cell cultures lacking the multicellular complexity and intercellular communications that exist in real tissues. 3D organoids present unique opportunities to investigate the functions of circadian rhythms in multicellular systems in vitro. For example, one could investigate circadian rhythms in the organoids with altered cell compositions or organoids derived from patients&#39; diseased tissues. This protocol enables the utilization of a bioluminescence assay to investigate different aspects of circadian rhythms in a more physiologically relevant in vitro model, organoids, which will help us better understand the roles of circadian rhythms in peripheral organs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 x 10 Falcon tissue culture dishes</td>
			<td>Fisher Scientific</td>
			<td>08-772A</td>
			<td></td>
		</tr>
		<tr>
			<td>A 83-01</td>
			<td>Sigma Aldrich</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Life Technologies</td>
			<td>12634-028</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50x)</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Micro-Fine IV Insulin Syringes</td>
			<td>Fisher Scientific</td>
			<td>14-829-1Bb</td>
			<td>Mfrn: BD 329424</td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Cayman Chemical</td>
			<td>13122</td>
			<td>GSK-3 inhibitor</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma Aldrich</td>
			<td>D4902-500MG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin (potassium salt)</td>
			<td>Cayman Chemical</td>
			<td>14681</td>
			<td></td>
		</tr>
		<tr>
			<td>Gastrin I Human</td>
			<td>Sigma Aldrich</td>
			<td>G9020</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth Factor reduced (GFR) Matrigel</td>
			<td>Corning</td>
			<td>CB-40230C</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>IntestiCult Organoid Growth Medium (Human)</td>
			<td>Stemcell Technologies</td>
			<td>06010</td>
			<td>Consist of IntestiCult OGM Human Basal Medium, 50 mL and Organoid Supplement, 50 mL. Mix both as 1:1 ratio to use as intestinal organoid growth medium</td>
		</tr>
		<tr>
			<td>Kronos Dio Luminometer Machine</td>
			<td>ATTO Corporation</td>
			<td>AB-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100x)</td>
			<td>Gibco</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>pABpuro-BluF reporter plasmid</td>
			<td>Addgene</td>
			<td>46824</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS without Calcium and Magnesium</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine EGF</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>R&#38;D Systems</td>
			<td>1254/10</td>
			<td>ROCK inhibitor</td>
		</tr>
	</tbody>
</table>

monitoring circadian oscillations with a bioluminescence reporter in organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

Bioluminescence recording was conducted to assess the circadian rhythmicity of human intestinal enteroids (HIEs) under two distinct conditions: stem cell-enriched conditions using intestinal organoid growth medium (Figure 3) versus differentiation-inducing conditions, which was achieved by replacing the intestinal organoid growth medium with a differentiation medium. On the day of the experiment, we synchronized the circadian clocks by performing a 1 h treatment with 100 nM Dexamethasone. Su...

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Author Spotlight: In Vitro Investigations of Circadian Rhythms in Multicellular Systems

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

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905 Views.  University of Cincinnati College of Medicine. Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Video: Author Spotlight: In Vitro Investigations of Circadian Rhythms in Multicellular Systems

Monitoring Actin Disassembly with Time-lapse Microscopy

Okay, so what I'd like to do today is to show you how to construct a flow chamber, a fusion chamber for imaging on an upright microscope. What then I then do is I create two spaces using film. So what I do is I take the para film, I cut it into small strips.Next I'm going to, this will basically form the channels for the, the walls, and I dry them with a tilted air. I put the cover slip on top of the para, And here's the finished. Then I heat it on a heat block to melt the param and basically create a seal.So what this is, is the, the cover glass here, and then on top of the cover glass is two layers of phim as you see right here. And then on top of this, again, there's a, it's a 22 by two two mil meter cover slip. This one right here, which I pressed against the param to fall the seal.Okay, so the liquid goes in in one direction here and I can suck it out with a filter paper here in this direction. And I'll show you how to do that in a bit. So what I like to do is I like to put it in a humidified chamber, which is very simple.Take two kind of elevated strips and have a filter of paper underneath. And to keep it noisy as I, I put the cover, cover slip on the flow chamber on. Now the way you introduce a solution to the chamber is that you just put the pipet tip at the entrance of the chamber and you just split.Now what I do is I incubate the chamber with the lid closed for 10 minutes. What I usually do to suck the liquid out from the other side is to use it, get a filter paper and cut it into thin strips like this. And I take one of these thin strips and I simultaneously apply the solution into the entrance while putting the filter paper at the exit to suck the exchange, the solution.So you'll see the liquid flowing through the solution. And then I wait for five minutes for the mid close. Now what I'm ready to do is to actually put that the actin it and the actin, I actually have to polymerize, I polymerize inside the flow chamber and it's just the same procedures before.So because like this, and then now that the action is polymerized, I wash out all the un excess un polymerized action using a large volume of just buffer. So here I have about a hundred microliters and it's a few reaction chamber volumes worth of buffer. Just, you know, when the filter paper becomes saturated, I have to change the direction.Okay?And so I'm ready to do imaging with this. So perfusion chamber with actin attached to it. Now I take the slide, mount it under the microscope, and since I'm imaging with an oil objective, I'm gonna put some immersion oil on the top surface of the cover, grass touch, such so assess solution or protein or anything on acting inside.So what I do is I can actually exchange the solution side while I'm doing the time lapse imaging. As such.

Monitoring Actin Demontaż z poklatkowy Mikroskopia

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Functional Imaging with Reinforcement, Eyetracking, and Physiological Monitoring

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, simple perceptual tasks are combined with appetitive and aversive reinforcement.  However, the use of reinforcers such as juice and airpuffs can create challenges for fMRI.  Reinforcer delivery can cause head movement, which creates artifacts in the fMRI signal.  Reinforcement can also lead to changes in heart rate and respiration that are mediated by autonomic pathways.  Changes in heart rate and respiration can directly affect the fMRI (BOLD) signal in the brain and can be confounded with signal changes that are due to neural activity.  In this presentation, we demonstrate methods for administering reinforcers in a controlled manner, for stabilizing the head, and for measuring pulse and respiration.

This presentation demonstrates the use of fMRI to study neural circuits that underlie decision-making. Simple perceptual tasks are combined with appetitive and aversive reinforcements to investigate how outcomes affect decision processes.

We use functional brain imaging (fMRI) to study neural circuits that underlie decision-making.  To understand how outcomes affect decision processes, ...

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...