We are grateful to our colleagues from the University of Geneva: Jacques Philippe for constructive comments on this work, Ueli Schibler for invaluable help with the development of the perifusion system and for scientific inspiration, Andr&#233; Liani for the having conceived design, manufacturing and commissioning of the perfusion system, Lesa-Technology LTD company for the assistance in the perifusion system and Drip-biolumicorder software development, George Severi for assistance with the perifusion experiments, Ursula Loizides-Mangold for critically reading the manuscript, and Anne-Marie Makhlouf for lentivirus preparations; to Etienne Lefai, St&#233;phanie Chanon and Hubert Vidal (INSERM, Lyon) for preparing human primary myoblasts; and to Domenico Bosco and Thierry Berney (Human Islet Transplantation Center, Geneva University Hospital) for providing human islets. This work was funded by the Swiss National Science Foundation Grant No. 31003A_146475/1, the Sinergia Swiss National Science Foundation Grant No. CRSII3-154405, Fondation Romande pour la Recherche sur Diab&#232;te, Bo Hjelt Foundation, Fondation Ernst et Lucie Schmidheiny, and Soci&#233;t&#233; Acad&#233;mique de Gen&#232;ve (CD).

Ethics statement: Manipulations included in this protocol were approved by the Ethics Committee of the Geneva University Hospital and by the Ethical Committee SUD EST IV (Agreement 12/111) 19. Human islets were isolated from pancreases of brain-dead multi-organ donors in the Islet Transplantation Centre at the University Hospital of Geneva (Switzerland) as described by us in references 16,18, or obtained from a commercial source.
1. Preparation of Primary Cell Culture
<ol>
	<li>Human Pancreatic Islet Isolation, Dissociation and Culture 
	NOTE: Coat every tube, plastic tip or pipette with Connaught Medical Research Laboratories (CMRL) medium in order to prevent islets or islet cells from sticking to the plastic surface, which can lead to a significant loss of cell material.
	<ol>
		<li>One day prior to islet cell dissociation add 1 ml of laminin-5-rich extracellular matrix (derived from 804G cells as described in reference 29) per 3.5 cm dish. Before plating cells, aspirate the matrix and wash the dish 3 times with sterile bi-distillated water. Allow the dish to dry under the laminar flow cabinet for 5 min.</li>
		<li>Inside the laminar flow cabinet, distribute the obtained islets with CMRL medium into 15 ml tube(s). Centrifuge at 272 x g for 5 min.</li>
		<li>Aspirate the supernatant, and then resuspend the pellet in 1 ml of sterile Dulbecco&#39;s phosphate-buffered saline (DPBS) pre-warmed to 37 &#176;C without calcium and magnesium. Centrifuge at 272 x g for 5 min.</li>
		<li>Aspirate the supernatant and resuspend the cell pellet in 1 ml of DPBS.</li>
		<li>To count the total number of islets, pipet 10 &#181;l from the 1 ml islet suspension into a new 3.5 cm dish. Count the number of islets in the 10 &#181;l drop under the microscope and from this calculate the total number of islets in the 1 ml islet suspension. Add 14 ml of DPBS and centrifuge the cell suspension one more time at 272 x g for 5 min.</li>
		<li>For islet cell dissociation, aspirate the supernatant and add 1 ml of cell detachment solution for a maximum of 1,000 islet equivalents (IEQ). Place the tube in a water bath at 37 &#176;C and gently mix the islets by pipetting up and down several times every minute, during 6-10 min. 
		NOTE: To check the digestion quality, pipet a 2 &#181;l drop of suspension on a glass slide and check under the microscope that all cells are well separated, and that no doublets or cell clumps have remained.</li>
		<li>Stop the reaction by adding 14 ml of cold CMRL with supplements (10% fetal bovine serum (FBS), 1% of L-alanyl-L-glutamine dipeptide, 1% penicillin-streptomycin (P/S), 1% gentamycin, 1% sodium pyruvate). Centrifuge at 425 x g for 5 min. Aspirate the supernatant and add 15 ml of CMRL medium to the cell pellet.</li>
		<li>Resuspend the pellet in a small volume of CMRL with supplements. Count the number of cells under the microscope using a hemocytometer, adjust the CMRL volume in order to obtain a cell concentration of ~650, 000 cells/ml.</li>
		<li>Pipet 3 separated drops of 100 &#181;l each from the dispersed cell suspension obtained in step 1.1.8 in a 3.5 cm dish pre-coated with laminin. 
		NOTE: Cells attach to the dish in about 24 hr.</li>
		<li>Incubate cells (Figure 1A) in a tissue culture incubator at 37 &#176;C in a humid chamber. Change the medium of the cell drops every 2-3 days by aspirating 100 &#181;l from each drop and replacing it with the same volume of fresh medium.</li>
	</ol>
	</li>
	<li>Human Primary Myoblast Culture and Differentiation into Myotubes
	<ol>
		<li>Tissue biopsy, satellite cell isolation and myoblast culture 
		Note: Muscle biopsies were obtained from the group of Etienne Lefai (INSERM, Lyon, France) 19.
		<ol>
			<li>Purify primary skeletal myoblasts according to the previously described procedure 30.</li>
		</ol>
		</li>
		<li>Differentiating primary human myoblasts into myotubes
		<ol>
			<li>Take one vial (1 x 106 cells) of human myoblasts stored in liquid nitrogen and thaw cells quickly by putting the vial for 30 sec to 1 min in a water bath at 37 &#176;C with agitation.</li>
			<li>Pipet cells (1 ml) into 24 ml of growth medium composed of HAM F-10 supplemented with 20% FBS, 1% P/S, 0.5% gentamycin and 0.2% amphotericin B.</li>
			<li>Centrifuge 5 min at 150 x g.</li>
			<li>Remove the supernatant and resuspend cells with 15 ml of fresh growth medium per 2.5 x 105 cells.</li>
			<li>Plate at least 2.5 x 105 cells per F75 adherent flask. Keep the myoblasts in a cell incubator at 37 &#176;C and 5% CO2.</li>
			<li>Once cells reach 60-80% confluency, dissociate cells with trypsin-EDTA 0.05% for 1-2 min and plate them in 2 ml of growth medium on adherent 3.5 cm petri dishes.</li>
			<li>After reaching confluence, remove the growth medium.</li>
			<li>Start the differentiation protocol of human myoblasts into myotubes by culturing them in 2 ml of Dulbecco&#39;s Modified Eagle Medium (DMEM) containing 1 g/L of glucose, 2% FBS, 1% P/S, 0.5% gentamycin and 0.2% amphotericin B (differentiation medium) in a cell incubator at 37 &#176;C and 5% CO2. Change the medium every 2 to 3 days. 
			NOTE: Myotubes are usually formed within 7-10 days.</li>
			<li>Check muscle cell differentiation under the microscope (Figure 2A) by observing the fusion of myoblasts into polynucleated myotubes 19.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Small Interfering RNA (siRNA) Transfection
<ol>
	<li>siRNA Transfection of Human Islet Cells 
	NOTE: The transfection protocol is performed in drops of 100 &#181;l on the next day after cell dissociation (steps 1.1.1-1.1.10).
	<ol>
		<li>Aspirate 100 &#181;l of CMRL medium from each drop and replace it with the same volume of serum-free Minimal Essential Medium (MEM) 2 hr before transfection by pipetting.</li>
		<li>Prepare a MEM-based mix of transfection reagent and 50 nM of target siRNA (siClock) or 50 nM of non-targeting siControl according to the manufacturer&#39;s instructions.
		<ol>
			<li>For one dish with 3 drops prepare two 1.5 ml tubes with 200 &#181;l of MEM each.</li>
			<li>Add to one of these tubes 4 &#181;l of transfection reagent.</li>
			<li>Add to the second tube 1 &#181;l of the of appropriate siRNA stock solution (20 &#181;M).</li>
			<li>Agitate these two tubes slowly on the orbital shaker for 5 min and then mix the content of the tubes together and agitate for 20 more min.</li>
		</ol>
		</li>
		<li>Aspirate 100 &#181;l of MEM from each drop and replace it with the same volume of transfection mix obtained in the previous step by pipetting.</li>
		<li>Replace the transfection solution with CMRL medium after 4 hr of incubation at 37 &#176;C. Repeat steps 2.1.1-2.1.3 the following day for cell re-transfection.</li>
	</ol>
	</li>
	<li>siRNA Transfection of Human Myotubes
	<ol>
		<li>Before transfection, replace the medium (see step 1.2.2.8) with 2 ml of fresh differentiation medium per 3.5 cm petri dish.</li>
		<li>In a sterile 1.5 ml tube, prepare a mix of 20 nM siRNA (siControl or siClock), which corresponds to 2.4 &#181;l of a 20 &#181;M siRNA solution, and 12 &#181;l of transfection reagent diluted in 100 &#181;l of differentiation medium. Incubate the solution at room temperature for 15 min with gentle agitation.</li>
		<li>Transfect cells with 114.4 &#181;l of the siRNA mix per 3.5 cm petri dish and place cells into a tissue culture incubator at 37 &#176;C and 5% CO2 for 24 hr.</li>
	</ol>
	</li>
</ol>
3. Continuous Long-term Circadian Bioluminescence Recording Performed in Parallel with the Assessment of Hormone Secretion in Living Human Primary Cells
<ol>
	<li>Introducing Circadian Bioluminescence Reporters into Human Primary Cells by Lentiviral Transduction 
	NOTE: All procedures with lentiviral particles must be performed in a biosafety level 2 facility to take additional precautions for work with agents that pose a moderate potential hazard to personnel and the environment.
	<ol>
		<li>Prepare reporter lentiviral particles by co-transfecting the vector of interest pLenti6.4/R4R2/V5-DEST/Bmal1-luc or pLV156-Per2-dLuc (called Bmal1-luc and Per2-luc, respectively,) plasmid 31 with lentiviral vectors pMD2G and psPAX into 293T cells using the polyethylenimine method (for detailed procedure see reference 16).</li>
		<li>Titrate the obtained lentiviral particles (details on the titration can be obtained at http://lentilab.unige.ch/). For further experiments, use lentiviruses with titers ranging 104 to 105 transducing units [TU/&#181;l].</li>
		<li>Place dishes with human islet cells or human myoblasts (at 30-50% confluency) inside the laminar flow cabinet and replace the medium with 2 ml of fresh supplemented CMRL medium (see step 1.1.7) or growth medium (see step 1.2.2.2), respectively.</li>
		<li>Calculate the multiplicity of infection (MOI) (i.e. infectious particles (transducing units)/number of cells).</li>
		<li>Transduce the primary cell culture by pipetting lentivirus solution to the dish in order to obtain a MOI = 3 (for example, for 65,000 attached cells add 3 &#181;l of the virus solution with the titer of 6.5 x 104 to 100 &#181;l medium drop).</li>
		<li>Incubate overnight in a tissue culture incubator. Change medium the next day. 
		NOTE: Transduce human islet cells at least 4 days prior to bioluminescence recording in order to achieve sufficient expression of the reporter construct. Myoblasts are transduced during the expansion phase, then grown to confluence, and subsequently differentiated into myotubes.</li>
	</ol>
	</li>
	<li>In Vitro Synchronization of Human Primary cells
	<ol>
		<li>Add 10 &#181;M of adenylyl cyclase activator in 2 ml of medium per 3.5 cm petri dish containing the primary cells previously transduced in step 3.1.5.</li>
		<li>Incubate for 60 min at 37 &#176;C in a cell culture incubator.</li>
		<li>Change the medium containing the adenylyl cyclase activator with 2.5 ml of the recording medium containing 100 &#181;M luciferin. 
		NOTE: For human islet cells use CMRL supplemented with 10% FBS, 1% L-alanyl-L-glutamine dipeptide, 1% P/S, 1% gentamycin; for human myotubes use phenol red - free DMEM with 1 g/L glucose supplemented with 2% FBS, 2% L-alanyl-L-glutamine dipeptide, 1% P/S, 0.5% gentamycin and 0.2% amphotericin B.</li>
	</ol>
	</li>
</ol>
4. Parallel Assessment of Circadian Bioluminescence Recording and Hormone Secretion Profiles in Synchronized Human Secretory Primary Cells
<ol>
	<li>Setting Up Long-term Constant Perifusion and Bioluminescence Recording for Human Primary Cells. 
	NOTE: When working outside the laminar flow cabinet, clean all contact surfaces and limit exposure of cultures or medium to the air to avoid contamination.
	<ol>
		<li>To prepare the perifusion medium, add 100 &#181;M of luciferin to the medium. 
		NOTE: For human islet cells use CMRL supplemented with 10% FBS, 1% L-alanyl-L-glutamine dipeptide, 1% P/S, 1% gentamycin; for human myotubes use phenol red &#8211; free DMEM with 1 g/L glucose supplemented with 2% FBS, 2% L-alanyl-L-glutamine dipeptide, 1% P/S, 0.5% gentamycin and 0.2% amphotericin B.</li>
		<li>Inside the laminar flow cabinet, open the 3.5 cm dishes containing the transduced, transfected and synchronized primary cell cultures (islet cells or myotubes) as described above. Insert sterile metallic caps (developed in house) (Figure 1B2) into the 3.5 cm dishes that are equipped with silicone influx/efflux connecting tubes (Figure 1B1/B5).</li>
		<li>Place the dishes on the measurement platform in the 37 &#176;C light-tight incubator. Fix the dishes to the platform by using a screwable adaptor (Figure 1B3). Insert the influx/efflux tubes of the perifusion system into the appropriate silicone tubes of the cap (Figure 1B1/B5) and set the speed of the pump at a flow rate of ~0.5 ml of medium per 1 hr.</li>
		<li>Open the in-house developed Drip-biolumicorder software that records the signals from the photomultiplier tube (PMT) detector. Chose the directory where the data will be stored and start continuous bioluminescence recording from each dish by clicking the &#34;start&#34; icon. 
		NOTE: Alternatively to the Drip-biolumicorder software, other programs (e.g. LumiCycle), can be used to record signals from PMT detector.</li>
		<li>Place sterile 6-well tissue culture plates in the collection box on ice.</li>
		<li>Open the control software that controls the timing of the automated switch among the collection wells. Set up the time window of medium collection (sec). Start collection of the outflow medium every 4 hr (14,400 sec; ~2 ml per time-point) by clicking the &#34;run&#34; icon.</li>
		<li>Transfer and measure the outflow medium from each collection well into sterile 2 ml tubes by pipetting. Keep tubes in a -20 &#176;C freezer before starting the next step. Repeat steps 4.1.5-4.1.6 every 24 hr.</li>
		<li>Stop the bioluminescence recording and the medium flow by clicking the &#34;stop&#34; icon on the corresponding software. Remove the metallic caps and aspirate the residual medium from the dishes.</li>
		<li>In order to normalize the secreted protein values obtained in different experiments, either extract DNA (normalization by genomic DNA content for myotubes 19), or add 1 ml of lysis acid-ethanol buffer (normalization by total hormone content for islet cells 18) to the dishes.</li>
	</ol>
	</li>
</ol>
5. Measuring Islet Hormone and Myokine Levels in the Outflow Medium Obtained by Continuous Perifusion of Human Primary Endocrine Cells
<ol>
	<li>Insulin
	<ol>
		<li>Quantify basal insulin levels in the outflow medium from collected time-points by using a human insulin enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer&#39;s instructions.</li>
		<li>Normalize data to the absolute volume of collected medium in each well and to the total insulin content, extracted from acid-ethanol treated cells at the end of the experiment (step 4.1.9) 18.</li>
	</ol>
	</li>
	<li>Interleukin-6 (IL-6)
	<ol>
		<li>Quantify&#160; basal IL-6 levels in the outflow medium from collected time-points by using a human IL-6 ELISA kit following the manufacturer&#39;s instructions. </li>
		<li>Normalize data to the absolute volume of collected medium in each well and to genomic DNA content at the end of the experiment (step 4.1.9).</li>
	</ol>
	</li>
</ol>
6. Circadian Dataset Analyses for Bioluminescence and Hormone Secretion Profiles
<ol>
	<li>Bioluminescence Analysis
	<ol>
		<li>Analyze bioluminescence profile using the provided software 19.</li>
	</ol>
	</li>
	<li>JTK-cycle Analysis
	<ol>
		<li>Analyze hormone secretion profiles and bioluminescence recording results by using the JTK_CYCLE algorithm 32.</li>
		<li>Set the circadian period width at 20-24 hr. 
		NOTE: In case the experimental conditions were recorded in parallel, a paired statistical analysis can be performed to compare the experiments.</li>
	</ol>
	</li>
	<li>CosinorJ Analysis
	<ol>
		<li>Alternatively, analyze hormone secretion and circadian bioluminescence profiles using the CosinorJ software 28.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Albrecht, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+to+perfection:+the+biology+of+central+and+peripheral+circadian+clocks.">Timing to perfection: the biology of central and peripheral circadian clocks.</a> Neuron. 74 (2), 246-260 (2012).</li><li>Dibner, C., Schibler, U., Albrecht, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+circadian+timing+system:+organization+and+coordination+of+central+and+peripheral+clocks.">The mammalian circadian timing system: organization and coordination of central and peripheral clocks.</a> Annu Rev Physiol. 72, 517-549 (2010).</li><li>Dibner, C., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+timing+of+metabolism+in+animal+models+and+humans.">Circadian timing of metabolism in animal models and humans.</a> J Intern Med. , (2015).</li><li>Marcheva, B., 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=Circadian+clocks+and+metabolism.">Circadian clocks and metabolism.</a> Handb Exp Pharmacol. (217), 127-155 (2013).</li><li>Philippe, J., Dibner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+circadian+timing:+roles+in+physiology+and+thyroid+malignancies.">Thyroid circadian timing: roles in physiology and thyroid malignancies.</a> J Biol Rhythms. 30 (2), 76-83 (2015).</li><li>Andrews, J. L., 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=CLOCK+and+BMAL1+regulate+MyoD+and+are+necessary+for+maintenance+of+skeletal+muscle+phenotype+and+function.">CLOCK and BMAL1 regulate MyoD and are necessary for maintenance of skeletal muscle phenotype and function.</a> Proc Natl Acad Sci U S A. 107 (44), 19090-19095 (2010).</li><li>McCarthy, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+circadian+transcriptome+in+adult+mouse+skeletal+muscle.">Identification of the circadian transcriptome in adult mouse skeletal muscle.</a> Physiol Genomics. 31 (1), 86-95 (2007).</li><li>Shostak, A., Husse, J., Oster, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+regulation+of+adipose+function.">Circadian regulation of adipose function.</a> Adipocyte. 2 (4), 201-206 (2013).</li><li>Dallmann, R., Viola, A. U., Tarokh, L., Cajochen, C., Brown, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+circadian+metabolome.">The human circadian metabolome.</a> Proc Natl Acad Sci U S A. 109 (7), 2625-2629 (2012).</li><li>Adamovich, Y., 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=Circadian+clocks+and+feeding+time+regulate+the+oscillations+and+levels+of+hepatic+triglycerides.">Circadian clocks and feeding time regulate the oscillations and levels of hepatic triglycerides.</a> Cell Metab. 19 (2), 319-330 (2014).</li><li>Chua, E. C., 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=Extensive+diversity+in+circadian+regulation+of+plasma+lipids+and+evidence+for+different+circadian+metabolic+phenotypes+in+humans.">Extensive diversity in circadian regulation of plasma lipids and evidence for different circadian metabolic phenotypes in humans.</a> Proc Natl Acad Sci U S A. 110 (35), 14468-14473 (2013).</li><li>Kalsbeek, A., Fliers, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daily+regulation+of+hormone+profiles.">Daily regulation of hormone profiles.</a> Handb Exp Pharmacol. (217), 185-226 (2013).</li><li>Hastings, M., O'Neill, J. S., Maywood, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+clocks:+regulators+of+endocrine+and+metabolic+rhythms.">Circadian clocks: regulators of endocrine and metabolic rhythms.</a> J Endocrinol. 195 (2), 187-198 (2007).</li><li>Muhlbauer, E., Wolgast, S., Finckh, U., Peschke, D., Peschke, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indication+of+circadian+oscillations+in+the+rat+pancreas.">Indication of circadian oscillations in the rat pancreas.</a> FEBS Lett. 564 (1-2), 91-96 (2004).</li><li>Marcheva, B., 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=Disruption+of+the+clock+components+CLOCK+and+BMAL1+leads+to+hypoinsulinaemia+and+diabetes.">Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes.</a> Nature. 466 (7306), 627-631 (2010).</li><li>Pulimeno, P., 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=Autonomous+and+self-sustained+circadian+oscillators+displayed+in+human+islet+cells.">Autonomous and self-sustained circadian oscillators displayed in human islet cells.</a> Diabetologia. 56 (3), 497-507 (2013).</li><li>Perelis, M., 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=Pancreatic+beta+cell+enhancers+regulate+rhythmic+transcription+of+genes+controlling+insulin+secretion.">Pancreatic beta cell enhancers regulate rhythmic transcription of genes controlling insulin secretion.</a> Science. 350 (6261), (2015).</li><li>Saini, C., 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=A+functional+circadian+clock+is+required+for+proper+insulin+secretion+by+human+pancreatic+islet+cells.">A functional circadian clock is required for proper insulin secretion by human pancreatic islet cells.</a> Diabetes Obes Metab. , (2015).</li><li>Perrin, L., 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=Human+skeletal+myotubes+display+a+cell-autonomous+circadian+clock+implicated+in+basal+myokine+secretion.">Human skeletal myotubes display a cell-autonomous circadian clock implicated in basal myokine secretion.</a> Mol Metab. 4 (11), 834-845 (2015).</li><li>Saini, C., Brown, S. A., Dibner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+peripheral+clocks:+applications+for+studying+circadian+phenotypes+in+physiology+and+pathophysiology.">Human peripheral clocks: applications for studying circadian phenotypes in physiology and pathophysiology.</a> Front Neurol. 6, 95 (2015).</li><li>Brown, S. A., 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=Molecular+insights+into+human+daily+behavior.">Molecular insights into human daily behavior.</a> Proc Natl Acad Sci U S A. 105 (5), 1602-1607 (2008).</li><li>Asher, G., 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=SIRT1+regulates+circadian+clock+gene+expression+through+PER2+deacetylation.">SIRT1 regulates circadian clock gene expression through PER2 deacetylation.</a> Cell. 134 (2), 317-328 (2008).</li><li>Dibner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+robustness+of+mammalian+circadian+oscillators.">On the robustness of mammalian circadian oscillators.</a> Cell Cycle. 8 (5), 681-682 (2009).</li><li>Dibner, C., 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=Circadian+gene+expression+is+resilient+to+large+fluctuations+in+overall+transcription+rates.">Circadian gene expression is resilient to large fluctuations in overall transcription rates.</a> EMBO J. 28 (2), 123-134 (2009).</li><li>Nagoshi, E., 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=Circadian+gene+expression+in+individual+fibroblasts:+cell-autonomous+and+self-sustained+oscillators+pass+time+to+daughter+cells.">Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells.</a> Cell. 119 (5), 693-705 (2004).</li><li>Sage, D., Unser, M., Salmon, P., Dibner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+software+solution+for+recording+circadian+oscillator+features+in+time-lapse+live+cell+microscopy.">A software solution for recording circadian oscillator features in time-lapse live cell microscopy.</a> Cell Div. 5, (2010).</li><li>Kowalska, E., Moriggi, E., Bauer, C., Dibner, C., Brown, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circadian+clock+starts+ticking+at+a+developmentally+early+stage.">The circadian clock starts ticking at a developmentally early stage.</a> J Biol Rhythms. 25 (6), 442-449 (2010).</li><li>Mannic, T., 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=Circadian+clock+characteristics+are+altered+in+human+thyroid+malignant+nodules.">Circadian clock characteristics are altered in human thyroid malignant nodules.</a> J Clin Endocrinol Metab. 98 (11), 4446-4456 (2013).</li><li>Parnaud, G., 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=Proliferation+of+sorted+human+and+rat+beta+cells.">Proliferation of sorted human and rat beta cells.</a> Diabetologia. 51 (1), 91-100 (2008).</li><li>Agley, C. C., Rowlerson, A. M., Velloso, C. P., Lazarus, N. L., Harridge, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+quantitative+immunocytochemical+characterization+of+primary+myogenic+cells+and+fibroblasts+from+human+skeletal+muscle.">Isolation and quantitative immunocytochemical characterization of primary myogenic cells and fibroblasts from human skeletal muscle.</a> J Vis Exp. (95), e52049 (2015).</li><li>Liu, A. C., 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=Redundant+function+of+REV-ERBalpha+and+beta+and+non-essential+role+for+Bmal1+cycling+in+transcriptional+regulation+of+intracellular+circadian+rhythms.">Redundant function of REV-ERBalpha and beta and non-essential role for Bmal1 cycling in transcriptional regulation of intracellular circadian rhythms.</a> PLoS Genet. 4 (2), e1000023 (2008).</li><li>Hughes, M. E., Hogenesch, J. B., Kornacker, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JTK_CYCLE:+an+efficient+nonparametric+algorithm+for+detecting+rhythmic+components+in+genome-scale+data+sets.">JTK_CYCLE: an efficient nonparametric algorithm for detecting rhythmic components in genome-scale data sets.</a> J Biol Rhythms. 25 (5), 372-380 (2010).</li><li>Dyar, K. A., 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=Muscle+insulin+sensitivity+and+glucose+metabolism+are+controlled+by+the+intrinsic+muscle+clock.">Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock.</a> Mol Metab. 3 (1), 29-41 (2014).</li><li>Innominato, P. F., 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=The+circadian+timing+system+in+clinical+oncology.">The circadian timing system in clinical oncology.</a> Ann Med. 46 (4), 191-207 (2014).</li><li>Chitikova, Z., 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=Identification+of+new+biomarkers+for+human+papillary+thyroid+carcinoma+employing+NanoString+analysis.">Identification of new biomarkers for human papillary thyroid carcinoma employing NanoString analysis.</a> Oncotarget. 6 (13), 10978-10993 (2015).</li><li>Pagani, L., 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=The+physiological+period+length+of+the+human+circadian+clock+in+vivo+is+directly+proportional+to+period+in+human+fibroblasts.">The physiological period length of the human circadian clock in vivo is directly proportional to period in human fibroblasts.</a> PLoS One. 5 (10), e13376 (2010).</li></ol>

The authors have nothing to disclose.

The experimental settings described here are composed of lentiviral delivery of circadian bioluminescence reporters into cultured human primary cells, followed by subsequent in vitro synchronization and continuous recording of bioluminescence for several days, and parallel analysis of hormone secretion by the same cells. They represent an efficient approach for exploring molecular mechanisms and functional aspects of circadian clocks in human primary cells.
The quality of the donor material is an important issue for the preparation of viable primary tissue cultures. The quality of human islets should be evaluated each time before starting the experiment. Islets with estimated purity or/and viability inferior to 70% are not recommended for these experiments. Islet cells tend to re-establish contacts in dissociated cultures, which plays an important role in their survival and function. Since islet cells do not proliferate in culture, they must be plated at a high density, which allows cells to establish contacts with neighboring cells. This is achieved by plating cells in droplets of a small volume. Importantly, cell death is higher in low-density islet cell cultures. Note that medium replacement should be performed promptly in order to avoid cell drying.
Myoblasts should be passaged preferably at 60% confluence, since a higher density may induce myoblast differentiation. After trypsinization, myoblasts should be carefully resuspended and dispersed to avoid cell clusters.
Bacterial or fungal contamination of primary cells should be excluded microscopically before starting the perifusion assay. Culture medium might be supplemented with antifungal substances. Additionally, the perifusion tubing should be rinsed by alcohol iodine disinfection/sterile water and the metallic caps should be sterilized by autoclaving. These steps are recommended between each experiment.
For efficient bioluminescence recording, the quality of the reporter lentivirus preparation should be determined by the intensity of the bioluminescent signal. The details of lentivirus production including troubleshooting can be found at http://lentilab.unige.ch/. Prior to plasmid transfection for lentivirus production, 293T cells should be plated at 30-50% confluence. It is highly recommended to perform a control of the transfection efficiency in a parallel dish, for instance by using CMV-GFP or an alternative fluorescent lentivector. For each virus preparation the virus titer should be established.
As the described experiments are typically long-lasting (48 hr or more), it is crucial to have stable silencing during this time span. The concentration of siRNA should be optimized as well as the cell confluency according to the siRNA reagent protocol. Efficiency of gene silencing must be tested at the end of the experiment by RT-qPCR or by Western blotting.
During perifusion, the flow rate determines the time necessary to completely exchange the medium in the dish but also has a mechanical impact on the primary cell culture. Indeed, setting up the flow at a rate, which is too low, will not allow for a complete exchange of medium in the dish, and will decrease the sensitivity of the method. On the contrary, a high-speed flow rate may damage the cells. In our hands, the optimal speed for both cell types did allow to collect 0.5 ml of the outflow medium per 1 hr.
Importantly, to obtain a measurable concentration of substances in the outflow medium, a sufficient number of secreting cells should be present in the perifused dish. The follow up method for the detection of the secreted substance (ELISA or other) should be sensitive enough, especially for substances secreted in very low concentrations. Higher cell density might be recommended to overcome this problem when possible. Alternatively, the outflow medium can be concentrated by dialysis or with centrifugal filters, as described by us in details in reference 19.
Taken together our experiments in human pancreatic islet cells and in primary myotubes provide for the first time compelling evidence that these human cells possess high-amplitude cell-autonomous circadian clocks 18-19. Employing the here described perifusion system combined with a luminometer device (Figure 1B) we have demonstrated that these clocks play an important role in the circadian regulation of basal insulin secretion by pancreatic islet cells (Figure 1D), and of basal IL6 secretion by skeletal myotubes (Figure 2C). Moreover, by knocking down the core clock gene CLOCK in both experimental systems, we show that a functional circadian oscillator is required for proper rhythmic insulin and IL-6 secretion by human islet and skeletal muscle cells, respectively (Figures 1C, D and 2B, C). Our results indicate a critical role of the human islet clock for proper insulin secretion, and are in good agreement with works performed in rodent genetic models 15,17. Given the major role of the circadian clock in allowing organisms to anticipate daily environmental changes rather than to react to them, circadian regulation of basal insulin and myokine secretion might represent such an anticipatory mechanism that coordinates pancreatic islet and skeletal muscle secretory activities to the rest-activity cycle of the whole body.
The here proposed methodology can be easily modified in order to study additional hormone secretion from the same tissues. We have already analyzed an additional large panel of myokines secreted by skeletal muscle cells, using multiplex human myokine arrays 19, and we are in the process of assessing glucagon secretion by human islet cells using the Glucagon ELISA kit (data not shown). Moreover, these experimental conditions can be optimized for other cell types, for instance primary adipocytes, for studying adipokine secretion, primary thyrocytes for studying thyroid hormone secretion, primary enterocytes for studying incretins secretion, etc. An additional important application of this system could be to explore the impact of physiological and/or pharmacological compounds on cellular circadian clock function and secretion. The compound of interest might be applied continuously throughout the entire experiment (for instance studying insulin secretion by human islet cells in the presence of high glucose or different levels of free fatty acids), or the compound might be added at a chosen phase of the circadian cycle.
This technique has the limitations of an in vitro study and does not represent the complexity of circadian rhythm regulation on a whole body level. At the same time, it helps to distinguish the role of an autonomous clock on cellular metabolism under controlled conditions. Currently available methods are based on snapshot measurements of secretion activity in the cells or explants following in vitro synchronization 17. The protocol described here represents a unique method allowing a concordant and continuous analysis of circadian rhythm and secretory activity within the same cell culture. Alternatively, this methodology could be applied to study the kinetics of non-circadian bioluminescent reporters, in conjunction with cell secretion, as well as for detecting the effects of different substances added to the perifusion medium on cell function. The critical steps in the protocol are: 1) efficient lentivirus preparation, 2) efficient cell transfection with siRNA and reporter vectors transduction; 3) constant medium outflow collection and 4) making sure that a sufficient number of cells is used in order to obtain measurable levels of hormones or cytokines in the collected outflow medium (see the troubleshooting above).
In view of recent evidences on the link between circadian clock perturbations and metabolic diseases and cancer in humans 3-4,28,34-35, studying human peripheral clock properties in primary cultures may represent an important and unique approach for understanding the potential clock connection to these diseases. Thus, our discovery of the existence of links between functional human pancreatic islet and skeletal muscle clock and basal secretion of insulin and myokines, might bear potential consequences for the understanding of the development of chronic diseases, such as obesity and type 2 diabetes 18-19 and will bring new avenues in the treatment of these diseases. Importantly, due to the established correlation between the in vitro assessed oscillator characteristics and the in vivo circadian phenotype 36, implication of human circadian clock properties as a hallmark of diseases, is of highest and immediate clinical relevance 20.

The circadian timing system (from Latin &#34;Circa diem&#34;) has emerged in all light-sensitive organisms, as an adaptive mechanism to the rotation of the Earth. In mammals, it is organized in a hierarchical manner, encompassing the central clock, which is situated in the suprachiasmatic nucleus of the ventral hypothalamus, and peripheral (or slave) oscillators that are operative in different organs. Moreover, these cell autonomous self-sustained oscillators are functional in nearly every cell of the body 1. Photic signals represent a dominant synchronizing cue (Zeitgeber) for the SCN neurons, whereas neural and humoral signals emanating from the SCN reset the peripheral clocks. In addition rest-activity rhythms, that drive in turn feeding-fasting cycles, are further synchronizers for peripheral clocks 2. According to our current understanding, the molecular makeup of the core clock is based on transcriptional and translational feedback loops, which are conserved between organisms. This comprises the transcriptional activators BMAL1 and CLOCK, which together activate transcription of the negative core clock PER and CRY genes. High levels of PER and CRY proteins will inhibit their own transcription through inhibition of the BMAL1/CLOCK complex. An auxiliary loop consists of the nuclear receptors REV-ERBs and RORs, which also regulate the transcription of BMAL1 and CLOCK. Furthermore, posttranslational events including phosphorylation, sumoylation, acetylation, O-GlcNAcylation, degradation and nuclear entry of the core clock proteins represent an additional important regulatory layer in establishing the 24 hr oscillation cycle 3.
Accumulating evidence stems from studies in rodent models and highlights the critical role of the circadian system in the coordination of metabolic and endocrine functions 4-5. A number of large-scale transcriptome analysis suggest that feeding &#8211; fasting cycles play a central role in the synchronization of peripheral oscillators 6-8. In an agreement with these studies, metabolomic and lipidomic analysis in rodents and humans have revealed that a large number of metabolites oscillate in tissue, plasma, and saliva in a circadian manner 9-11. Importantly, most hormones exhibit circadian rhythms in blood 5,12-13. Moreover, circadian clocks of the corresponding hormone producing peripheral tissue might regulate hormone secretion locally. Cell-autonomous circadian oscillators have been described in rodent and human pancreatic islet cells 14-16. These oscillators play an essential role in regulating the pancreatic islet transcriptome and function 15,17-18. Furthermore, myokine secretion by human skeletal myotubes has been recently demonstrated to exhibit a circadian pattern, which is regulated by cell-autonomous oscillators operative in these cells 19.
Several approaches for studying circadian rhythms in humans in vivo have been widely used. For instance, plasma melatonin or cortisol levels as well as thoracic skin surface temperature (reviewed in references 3,20) have been studied to assess endogenous circadian clocks. Although these methods allow studying systemic circadian oscillations in vivo, they are far from providing a reliable assessment of free-running autonomous circadian rhythms in different organs and tissues. Nevertheless, such dissection from the systemic regulation would be an indispensable tool for understanding the specific effect of intracellular molecular clocks on the function of these cells. Therefore, a substantial effort has been undertaken to develop reliable approaches for studying human clocks in immortalized or primary cultured cells synchronized in vitro. Importantly, it has been demonstrated that clock characteristics measured in cultured primary skin fibroblast cells closely reflect the individual clock properties of the whole organism 21. The development of fluorescent and bioluminescent circadian reporters has greatly advanced this approach 22-27. Furthermore, studying primary cell clocks that are derived from different peripheral organs allows for the investigation of the molecular properties of human tissue-specific clocks 3,5,16,19-20,28. Thus, assessment of circadian clocks in in vitro synchronized primary explants or cells, by using bioluminescent reporters, represents a highly useful method to study the molecular makeup of human peripheral clocks and their impact on organ function.
In this article, we will present detailed protocols for assessing circadian gene expression in human primary islet and skeletal muscle cells synchronized in vitro as well as the impact of autonomous cellular clock disruption on the secretory function of these cells.

<table><tbody><tr><td>Trypsin-EDTA</td><td>Invitrogen</td><td>25300-054</td><td>For muscle biopsy digestion</td></tr><tr><td>DPBS no calcium no magnesium</td><td>Invitrogen</td><td>14190-094</td><td></td></tr><tr><td>HAM F-10</td><td>Invitrogen</td><td>41550-021</td><td>For myoblasts culture</td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10270</td><td>Supplement to culture medium</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P0781-100</td><td>Supplement to culture medium</td></tr><tr><td>Gentamycin</td><td>Axon&amp;nbsp;</td><td>A1492.0001</td><td>Supplement to culture medium</td></tr><tr><td>Fungizone</td><td>Invitrogen</td><td>15290-026</td><td>Amphotericin B, supplement to culture medium</td></tr><tr><td>DMEM 1g/L glucose + Na pyruvate + glutamax&amp;nbsp;</td><td>Invitrogen</td><td>21885-025</td><td>For myotubes culture</td></tr><tr><td>DMEM 1g/L glucose -Na Pyruvate - glutamax</td><td>Invitrogen</td><td>11880-028</td><td>Recording medium for LumiCycle</td></tr><tr><td>Glutamax</td><td>Invitrogen</td><td>35050-028</td><td>L-alanyl-L-glutamine dipeptide, supplement to recording medium</td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT-104</td><td>Cell detachment solution, for islet cell dissociation</td></tr><tr><td>CMRL</td><td>Gibco</td><td>21530-027</td><td>Culture medium for islet cells</td></tr><tr><td>Sodium Pyruvate</td><td>Gibco</td><td>11360-039</td><td>Supplement to culture medium</td></tr><tr><td>15 ml High-Clarity Polipropylene Conical Tube</td><td>Falcon</td><td>352096</td><td></td></tr><tr><td>F75 flask</td><td>BD Falcon</td><td>353136</td><td></td></tr><tr><td>3.5 cm Petri dish&amp;nbsp;</td><td>BD Falcon</td><td>353001</td><td></td></tr><tr><td>Foskolin</td><td>Sigma</td><td>F6886</td><td>Adenylyl cyclase activator, used for synchronization</td></tr><tr><td>Luciferin</td><td>Prolume LTD</td><td>260150</td><td>Supplement to recording medium</td></tr><tr><td>OptiMEM&amp;nbsp;</td><td>Invitrogen</td><td>51985-026</td><td>Serum-free Minimal Essential Medium (MEM) used for human islet cells transfection</td></tr><tr><td>Lipofectamine RNAiMAX reagent</td><td>Invitrogen</td><td>13778-150</td><td>Transfection reagent</td></tr><tr><td>HiPerFect reagent</td><td>Qiagen</td><td>301705</td><td>Transfection reagent</td></tr><tr><td>ON-TARGET plus siCLOCK smartpool&amp;nbsp;</td><td>Dharmacon</td><td>L-008212-00</td><td></td></tr><tr><td>ON-TARGET plus non targeting siRNA #1 (siControl)</td><td>Dharmacon</td><td>D-001810-01</td><td></td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kit&amp;nbsp;</td><td>Qiagen</td><td>69504</td><td>For myotubes DNA extraction</td></tr><tr><td>RNeasy Plus Mini kit&amp;nbsp;</td><td>Qiagen</td><td>74104</td><td>For myotubes RNA extraction</td></tr><tr><td>QIAshredder&amp;nbsp;</td><td>Qiagen</td><td>79654</td><td>For myotubes RNA extraction</td></tr><tr><td>2 ml collecting tubes</td><td>Axygen</td><td>311-10-051</td><td>To collect the medium with the perifusion</td></tr><tr><td>Tissue culture Plate, 6 Well</td><td>BD Falcon&amp;nbsp;</td><td>353046</td><td>To collect the medium with the perifusion</td></tr><tr><td>RNeasy Plus Micro kit&amp;nbsp;</td><td>Qiagen</td><td>74034</td><td>For islet RNA extraction</td></tr><tr><td>Human IL-6 Instant ELISA kit&amp;nbsp;</td><td>eBioscience</td><td>88-7066-22</td><td></td></tr><tr><td>Human Insulin Kit Mercodia</td><td>Mercodia</td><td>10-1113-01</td><td></td></tr><tr><td>Hydrochloric acid, min,37%,p.a.</td><td>Acros organics</td><td>124630010</td><td>Used for preparation of lysis buffer (375 ml Ethanol + 7.5%HCl + 117.5% H&lt;sub&gt;2&lt;/sub&gt;O)</td></tr><tr><td>Ethanol (&gt;99.8%)</td><td>Fluka Analytical</td><td>02860-1L</td><td>Used for preparation of lysis buffer (375 ml Ethanol + 7.5%HCl + 117.5% H&lt;sub&gt;2&lt;/sub&gt;O)</td></tr><tr><td>Human Islets for Research</td><td>Prodo Laboratories</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Heraeus</td><td>Megafuge 1.0R</td><td></td></tr><tr><td>Water bath</td><td>VWR</td><td>1112A</td><td>&amp;nbsp;at 37 &amp;deg;C</td></tr><tr><td>Tissu culture hood</td><td>Faster&amp;nbsp;</td><td>SafeFastElite</td><td></td></tr><tr><td>Tissu culture incubator</td><td>Heraeus</td><td>HeraCell 150</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt; at 37 &amp;deg;C, no water due to the LumiCycle installation</td></tr><tr><td>Tissu culture incubator</td><td>Heraeus</td><td>HeraCell 150</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt; at 37 &amp;deg;C, no water due to the LumiCycle installation</td></tr><tr><td>Tissu culture incubator</td><td>Thermo Scientific</td><td>Hera Cell 150i</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt; at 37 &amp;deg;C</td></tr><tr><td>Shaker</td><td>Heidolph Instruments</td><td>Unimax 1010</td><td>For agitation of the siRNA mix</td></tr><tr><td>LumiCycle</td><td>Actimetrics</td><td></td><td></td></tr><tr><td>LumiCycle software</td><td>Actimetrics</td><td></td><td></td></tr><tr><td>CosinorJ software</td><td>EPFL</td><td></td><td>Freely available at: http://bigwww.epfl.ch/algorithms/cosinorj/</td></tr><tr><td>Rheodyne titan MX&amp;nbsp;</td><td>ERC GmbH</td><td></td><td>Control software that controls the timing of the automated switch</td></tr></tbody></table>

parallel measurement of circadian clock gene expression and hormone secretion in human primary cell cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Assessment of Islet Hormone Secretion with Parallel Circadian Bioluminescence Recording from Perifused Human Islet Cells
After providing a first molecular characterization of the circadian clock, operative in human islet cells 16, we aimed at studying the impact of clock disruption on islet function and transcription 18. We set up an efficient siClock transfection protocol in dispersed human islet cells (see Protocol for details), which resulted in more than 80% knockdown of CLOCK mRNA, and in efficient clock ablation as measured by circadian bioluminescence profiling 18. Glucose induced insulin secretion (GSIS) analysis revealed significantly reduced basal and stimulated insulin secretion upon such clock disruption (not shown). To assess hormone secretion by human islet cells around-the-clock, a continuous perifusion system was connected to a luminometer (depicted in Figure 1B). Human islet cells, bearing a functional (siControl) or dysfunctional (siClock) oscillator were transduced with Bmal1-luc lentiviral particles. Cells were subsequently synchronized with a pulse of adenylyl cyclase activator followed by parallel analysis of circadian bioluminescence and insulin secretion into the outflow medium during 48 hr (Figure 1C, D 18). These experiments suggest that under constant physiological glucose concentration (5.6 mM glucose), insulin secretion by in vitro synchronized human islet cells exhibits a circadian profile, which is disrupted in siClock bearing samples (Figure 1D).
Studying Myokine Secretion by Human Skeletal Myotubes Synchronized in vitro in the Presence or Absence of Functional Clock
In view of the potential role of the skeletal muscle clock in the regulation of glucose metabolism in rodents 33, we aimed at characterizing circadian rhythms in primary human skeletal myotubes and at investigating their role in myotube function 19. To this end, disruption of the circadian clock in skeletal myotubes was achieved by transfecting siRNA targeting CLOCK. Circadian bioluminescence reporter assays with Bmal1-luc and Per2-luc reporters revealed that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm, which was further confirmed by endogenous core clock transcript expression (Figure 2B; 19). This endogenous clock was efficiently disrupted in the presence of siRNA targeting CLOCK (Figure 2B). Moreover, the basal secretion of IL-6 (Figure 2C), interleukin-8 (IL-8) and Monocyte Chemotactic Protein 1 (MCP-1) (not shown) by synchronized skeletal myotubes, assessed by the here described perifusion system (Figure 1B) and subsequent large-scale myokine multiplex analysis (not shown), exhibits a circadian profile, which is strongly dysregulated upon clock disruption (Figure 2C 19).
<img alt="Figure 1" src="/files/ftp_upload/54673/54673fig1.jpg" /> 
Figure 1: Assessment of Hormone Secretion with Parallel Circadian Bioluminescence Recording from Perifused Human Islet Cells. (A) Representative picture of attached human islet cells one day after islet dissociation. (B) Schematic presentation of the home-made perifusion system that includes a bottle with the perifusion medium, a pump, a measurement platform equipped with a photomultiplier tube (PMT) within the light-tight incubator, a luminometer device, controlled by the recording software, and a semiautomatic sample collector. Insert: (B1) Influx connecting tube; (B2) metallic cap for the 3.5 cm Petri dish; (B3) screwable adaptor that attaches the cap to the measurement platform (B4); (B5) efflux connecting tube; (B6) automatically controlled medium distributor. (C) Human islet cells were transfected with either scrambled siRNA (siControl) or siRNA targeting CLOCK (siClock) and transduced with the Bmal1-luc reporter. Cells were constantly perifused with culture medium containing 5.6 mM glucose. Circadian bioluminescence was recorded following synchronization by an adenylyl cyclase activator pulse. (D) Insulin levels were assessed by ELISA in the outflow samples collected every 4 hr during 48 hr. Application of JTK_CYCLE algorithm 32 confirmed that in the presence of a functional clock (siControl), the average profile of secreted insulin was circadian within 48 hr, with a period length of 24.19 &#177; 0.89 hr (**p = 0.009; n = 7 donors). This circadian profile was lost upon clock disruption (siClock). Data are presented as % of secreted hormone from the total hormone content (mean &#177; SEM) for n = 7 donors (one replicate for each donor).This figure has been modified from reference 18. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54673/54673fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54673/54673fig2.jpg" /> 
Figure 2: Basal IL-6 Secretion by Human Skeletal Myotubes is Strongly Inhibited in the Absence of a Functional Circadian Clock. Myoblasts were transduced with lentiviral particles containing the Bmal1-luc transgene, differentiated into myotubes, and transfected with either siControl or siClock siRNA. 24 hr following transfection, myotubes were synchronized with an adenylyl cyclase activator pulse and subjected to continuous perifusion with parallel bioluminescence recording. (A) Representative pictures were taken at day 0 (D0) and D7 following the switch from 20% to 2% FBS containing medium to induce myotubes differentiation. During the differentiation process, human myoblasts are reoriented, become elongated and fuse together to form a plurinuclated syncytium. (B) Bmal1-luc bioluminescence profiles of siControl-transfected myotubes (black line) and siClock-transfected myotubes (grey line). Bmal1-luc oscillation profiles were recorded in three independent experiments (one donor per experiment). (C) Representative basal IL-6 secretion profile in the presence or absence of a functional clock. The perifusion outflow medium was collected in 4 hr intervals during 48 hr (0-4 corresponds to the accumulation of IL-6 between 0 hr and 4 hr). IL-6 levels in the medium were assessed by ELISA in two technical duplicates from three independent experiments. The results represent basal IL-6 levels normalized to the total DNA content. IL-6 secretion was reduced on average of 69.30 &#177; 10.61% upon circadian clock disruption (mean &#177; SEM, n = 3; *p &#60;0.05, paired t test). This figure has been modified from reference 19. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54673/54673fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures at JoVE.com

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

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Nanyang Technological University

Research

JoVE Journal

Genetics

8.2K Views.  University of Geneva Medical School, Institute of Genetics and Genomics in Geneva (iGE3). Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Video: Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Neuroscience

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse splenocytes with the pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS), ODN1826, and heat-killed Listeria monocytogenes and examine their effect on the molecular circadian clock. Previously, studies have focused on examining the influence of LPS on the molecular clock using a variety of in vivo and ex vivo approaches from an assortment of models (e.g., mouse, rat, and human). This protocol describes the isolation and challenge of splenocytes, as well as the methodology to assess clock gene expression post-challenge via quantitative PCR. This approach can be used to assess not only the influence of microbial components on the molecular clock but other molecules as well that may alter expression of the clock. This approach could be utilized to tease apart the molecular mechanism of how PAMP-Toll-like receptor interaction influences clock expression.

This protocol describes a technique using mouse splenocytes to discover pathogen-associated molecular patterns that alter molecular clock gene expression.

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

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 ...

Biology

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles.

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...

Bioengineering

Rapid Analysis of Circadian Phenotypes in Arabidopsis Protoplasts Transfected with a Luminescent Clock Reporter

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable biotic and abiotic stress. Conversely, disruption of circadian timekeeping severely compromises plant health and reduces agricultural crop yields. It is therefore imperative that we understand the intricate regulation of circadian rhythms in plants, including the factors that affect motion of the transcriptional clockwork itself.
Testing circadian defects in the model plant Arabidopsis thaliana (Arabidopsis) traditionally involves crossing specific mutant lines to a line rhythmically expressing firefly luciferase from a circadian clock gene promoter. This approach is laborious, time-consuming, and could be fruitless if a mutant has no circadian phenotype. The methodology presented here allows a rapid initial assessment of circadian phenotypes. Protoplasts derived from mutant and wild-type Arabidopsis are isolated, transfected with a rhythmically expressed luminescent reporter, and imaged under constant light conditions for 5 days. Luminescent traces will directly reveal whether the free-running period of mutant plants is different from wild-type plants. The advantage of the method is that any Arabidopsis line can efficiently be screened, without the need for generating a stably transgenic luminescent clock marker line in that mutant background.

The circadian clock regulates about a third of the Arabidopsis transcriptome, but the percentage of genes that feed back into timekeeping remains unknown. Here we visualize a method to rapidly assess circadian phenotypes in any mutant line of Arabidopsis using luminescent imaging of a circadian reporter transiently expressed in protoplasts.

The plant circadian clock allows the anticipation of daily changes to the environment. This anticipation aids the responses to temporally predictable ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

Optimizing Human Intestinal Enteroids for Circadian Rhythm Studies

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.

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.

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

Circadian Rhythms and Gene Regulation

The biological clock is involved in many aspects of regulating complex physiology in all animals. It was in 1935 when German zoologists, Hans Kalmus and Erwin B&#252;nning, discovered the existence of circadian rhythm in Drosophila melanogaster. However, the internal molecular mechanisms behind the circadian clock remained a mystery until 1984, when Jeffrey C. Hall, Michael Rosbash, and Michael W. Young discovered the expression of the Per gene oscillating over a 24-hour cycle. In subsequent years, many other associated genes were identified, and the mechanism of regulation of the circadian rhythms was further unraveled. The contribution of Jeffrey C. Hall, Michael Rosbash, and Michael W. Young to the understanding of the internal biological circadian clocks was recognized with the Nobel Prize for Physiology or Medicine in 2017.
<h4>The Molecular Mechanism of Circadian Rhythms</h4>
In Drosophila, the Period (PER) protein is the main regulatory protein that controls the internal circadian rhythms in cells. PER forms a complex with another essential protein called Timeless (TIM) and enters the nucleus. Here, it can regulate the levels of PER expression in the cell through feedback inhibition. Additionally, it also controls the expression of other genes by inhibiting the activity of the transcriptional activators Clock and Cycle. Importantly, the stability of the PER/TIM complex is dependent on the presence or absence of light, meaning it is degraded under daytime conditions. The result is that the expression of genes downstream of the PER/TIM complex is controlled by light, and this phenomenon allows for synchronization of the circadian clock.
In mammals, the regulation of the circadian rhythm works in a very similar manner. However, due to the addition of several paralog genes, the regulation of the entire pathway is much more complicated than in Drosophila.
<h4>Significance of Circadian Rhythms </h4>
All living organisms on Earth have evolved in the presence of 24-hour day/night cycles and adapted their cellular, physiological as well as behavioral responses accordingly. For example, the diurnal cycles of sleeping and waking, body temperature, and hormone release in mammals are controlled by the circadian rhythms. Irregular circadian rhythms can lead to many health issues, such as bipolar disorder or sleep disorders. Additionally, disruption of circadian rhythms can result in adverse effects on other systems of the body, including the cardiovascular system.

Circadian Rhythms and Per/Tim complex

The biological clock is involved in many aspects of regulating complex physiology in all animals. It was in 1935 when German zoologists, Hans Kalmus and ...

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

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