Name: measuring relative insulin secretion using a co-secreted luciferase surrogate
Uploaded: 2019-06-25T15:00:00
Description: Watch this Scientific Journal Video about Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate at JoVE.com

The authors thank all current and former members of the Cobb laboratory for valuable work and discussions, and Dionne Ware for administrative assistance. Michael Kalwat is supported by a Juvenile Diabetes Research Foundation SRA-2019-702-Q-R. This work was made possible through NIH R37 DK34128 and Welch Foundation Grant I1243 to Melanie Cobb. Early parts of this work were also supported by an NIH F32 DK100113 to Michael Kalwat.

1. Preparation of reagents, media and buffers (Table 1)
<ol>
	<li>Prepare MIN6 complete media in 500 mL of high-glucose (4.5 g/L) Dulbecco&#39;s modified Eagle medium (DMEM) with the following additives: 15% fetal bovine serum (FBS), 100 units/mL penicillin, 100 &#956;g/mL streptomycin, 292 &#956;g/mL L-glutamine, and 50 &#956;M &#946;-mercaptoethanol. 
	NOTE: The stable cell line in this case is maintained in 250 &#181;g/mL of G418 antibiotic.</li>
	<li>Prepare Krebs-Ringer bicarbonate buffer (KRBH) by making a s...

<ol>
	<li>Kalwat, M. 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=Insulin+promoter-driven+Gaussia+luciferase-based+insulin+secretion+biosensor+assay+for+discovery+of+beta-cell+glucose-sensing+pathways.">Insulin promoter-driven Gaussia luciferase-based insulin secretion biosensor assay for discovery of beta-cell glucose-sensing pathways.</a> ACS Sensors. 1 (10), 1208-1212 (2016).</li><li>Burns, S. 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=High-throughput+luminescent+reporter+of+insulin+secretion+for+discovering+regulators+of+pancreatic+Beta-cell+function.">High-throughput luminescent reporter of insulin secretion for discovering regulators of pancreatic Beta-cell function.</a> Cell Metabolism. 21 (1), 126-137 (2015).</li><li>Rajan, S., 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=In+vitro+processing+and+secretion+of+mutant+insulin+proteins+that+cause+permanent+neonatal+diabetes.">In vitro processing and secretion of mutant insulin proteins that cause permanent neonatal diabetes.</a> American Journal of Physiology - Endocrinology and Metabolism. 298 (3), E403-E410 (2010).</li><li>Watkins, S., 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=Imaging+Secretory+Vesicles+by+Fluorescent+Protein+Insertion+in+Propeptide+Rather+Than+Mature+Secreted+Peptide.">Imaging Secretory Vesicles by Fluorescent Protein Insertion in Propeptide Rather Than Mature Secreted Peptide.</a> Traffic. 3 (7), 461-471 (2002).</li><li>Welsh, J. 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S., 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=Connexin-36+Gap+Junctions+Regulate+In+Vivo+First-+and+Second-Phase+Insulin+Secretion+Dynamics+and+Glucose+Tolerance+in+the+Conscious.">Connexin-36 Gap Junctions Regulate In Vivo First- and Second-Phase Insulin Secretion Dynamics and Glucose Tolerance in the Conscious.</a> Diabetes. 61 (7), 1700-1707 (2012).</li><li>Benninger, R. K. P., Head, W. S., Zhang, M., Satin, L. S., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions+and+other+mechanisms+of+cell-cell+communication+regulate+basal+insulin+secretion+in+the+pancreatic+islet.">Gap junctions and other mechanisms of cell-cell communication regulate basal insulin secretion in the pancreatic islet.</a> The Journal of Physiology. 589 (22), 5453-5466 (2011).</li><li>Konstantinova, I., 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=EphA-Ephrin-A-mediated+beta+cell+communication+regulates+insulin+secretion+from+pancreatic+islets.">EphA-Ephrin-A-mediated beta cell communication regulates insulin secretion from pancreatic islets.</a> Cell. 129 (2), 359-370 (2007).</li><li>Jaques, 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=Dual+effect+of+cell-cell+contact+disruption+on+cytosolic+calcium+and+insulin+secretion.">Dual effect of cell-cell contact disruption on cytosolic calcium and insulin secretion.</a> Endocrinology. 149 (5), 2494-2505 (2008).</li><li>Calabrese, 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=Connexin+36+Controls+Synchronization+of+Ca2++Oscillations+and+Insulin+Secretion+in+MIN6+Cells.">Connexin 36 Controls Synchronization of Ca2+ Oscillations and Insulin Secretion in MIN6 Cells.</a> Diabetes. 52 (2), 417-424 (2003).</li><li>Bielefeld-Sevigny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AlphaLISA+immunoassay+platform-+the+&amp;#34;no-wash&amp;#34;+high-throughput+alternative+to+ELISA.">AlphaLISA immunoassay platform- the &amp;#34;no-wash&amp;#34; high-throughput alternative to ELISA.</a> Assay and Drug Development Technologies. 7 (1), 90-92 (2009).</li><li>Aslanoglou, D., George, E. 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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=Optical+tools+for+understanding+the+complexity+of+beta-cell+signalling+and+insulin+release.">Optical tools for understanding the complexity of beta-cell signalling and insulin release.</a> Nature Reviews Endocrinology. 14 (12), 721-737 (2018).</li><li>Zhu, S., 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=Monitoring+C-Peptide+Storage+and+Secretion+in+Islet+beta-Cells+In+Vitro+and+In+Vivo.">Monitoring C-Peptide Storage and Secretion in Islet beta-Cells In Vitro and In Vivo.</a> Diabetes. 65 (3), 699-709 (2016).</li></ol>

Herein we present a method to rapidly assess glucose-stimulated insulin secretion responses from MIN6 &#946; cells. For the best responses in the assay it is important to seed the MIN6 cells at the proper density and allow them to become 85-95% confluent. This improves &#946; cell responses to glucose because of improved cell-cell contacts and synchronization, which&#160; occurs both in primary islets17,18,19,<sup cla...

The method presented here allows insulin secretion from a genetically-modified beta cell line to be assayed rapidly and affordably in 96-well-plate format. The key to this protocol is a modified version of insulin with the naturally-secreted Gaussia luciferase (GLuc, ~18 kDa) inserted (see Figure 1) into the C-peptide to generate insulin-Gaussia (InsGLuc)1,2. Other larger proteins, such as GFP (~25 kDa), have been successfully inserted into the C-peptide of insulin and exhibited the expected post-translational processing from proinsulin-GFP to insulin and GFP-C-peptide3,4. For the assay in this protocol, GLuc has been codon-optimized for mammalian expression and two mutations have been introduced to enhance glow-like kinetics5,6. Multiple combinations and replicates of treatment conditions can be easily tested in 96-well-plate format and the secretion results can be obtained immediately following the experiment.
A major advantage, as previously noted2, is the low cost of this luciferase-based secretion measurement (&#60; $0.01/well) which differentiates it from the relatively higher costs and technical aspects of enzyme-linked immunosorbent assays (ELISAs) (&#62; $2/well) and homogenous time-resolved fluorescence (HTRF) or other F&#246;rster resonance energy transfer (FRET)-based antibody (&#62; $1/well) assays. In comparison to these antibody-based assays, which measure the concentration of insulin by referencing a standard curve, the InsGLuc assay measures secretory activity as a relative comparison to control wells on the plate. For that reason, every experiment requires the inclusion of proper controls. This distinction is a trade-off to allow rapid and inexpensive measurements. However, InsGLuc secretion has been demonstrated to be highly correlated with insulin secretion as measured by ELISA1,2. This technology has been scaled up for high-throughput screening1,2,7 and has led to the identification of novel modulators of insulin secretion including a voltage-gated potassium channel inhibitor7 as well as a natural product inhibitor of &#946; cell function, chromomycin A28. The use of InsGLuc is most appropriate for researchers who plan to continually test many different treatment conditions for their impact on insulin secretion. In follow-up experiments it is necessary to repeat key findings in a parental &#946; cell line, and optimally in murine or human islets, and measure insulin secretion using an antibody-based assay.

<table>
	<tbody>
		<tr>
			<td>Cell culture materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rIns-GLuc stable MIN6 cells</td>
			<td></td>
			<td></td>
			<td>Parental MIN6 cell line stably expressing pcDNA3.1+rInsp-Ins-eGLuc and maintained in 250 ug/ml G418</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td>4.5 g/L glucose media</td>
		</tr>
		<tr>
			<td>fetal bovine serum, heat-inactivated</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo-Fisher Scientific</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>beta-mercaptoethanol</td>
			<td>Thermo-Fisher Scientific</td>
			<td>BP 176-100</td>
			<td></td>
		</tr>
		<tr>
			<td>glutamine</td>
			<td>Thermo-Fisher Scientific</td>
			<td>BP379-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma</td>
			<td>T3924-500</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Gold Biotechnology</td>
			<td>G418-10</td>
			<td>Stock solution 250 mg/mL in water. Freeze aliquots at -20C.</td>
		</tr>
		<tr>
			<td>T75 tissue culture flasks</td>
			<td>Fisher Scientific</td>
			<td>07-202-000</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well tissue culture plates</td>
			<td>Celltreat</td>
			<td>229196</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent reservoirs (50 mL)</td>
			<td>Corning</td>
			<td>4870</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secretion assay reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BSA (RIA grade)</td>
			<td>Thermo-Fisher Scientific</td>
			<td>50-146-952</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma</td>
			<td>G8270-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Thermo-Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Thermo-Fisher Scientific</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes, pH 7.4</td>
			<td>Thermo-Fisher Scientific</td>
			<td>50-213-365</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Thermo-Fisher Scientific</td>
			<td>15568414</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Thermo-Fisher Scientific</td>
			<td>M9272-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C-7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Optional drugs for stimulation experiments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diazoxide</td>
			<td>Sigma</td>
			<td>D9035</td>
			<td>Stock solution: 50 mM in 0.1N NaOH. Add equal amount of 0.1N HCl to any buffer where diazoxide is added.</td>
		</tr>
		<tr>
			<td>epinephrine (bitartrate salt)</td>
			<td>Sigma</td>
			<td>E4375</td>
			<td>Stock solution: 5 mM in water</td>
		</tr>
		<tr>
			<td>PMA (phorbol 12-myristate)</td>
			<td>Sigma</td>
			<td>P1585</td>
			<td>Stock solution: 100 &#181;M in DMSO</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Guassia assay materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium phosphate (Na2HPO4)</td>
			<td>Thermo-Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Thermo-Fisher Scientific</td>
			<td>G334</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bromide</td>
			<td>Thermo-Fisher Scientific</td>
			<td>AC44680-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Thermo-Fisher Scientific</td>
			<td>AC44608-5000</td>
			<td>Stock solution: 0.5 M pH 8</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>RPI</td>
			<td>T60040-1000.0</td>
			<td>Stock solution: 1 M pH 8</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Fisher Scientific</td>
			<td>AAA1775922</td>
			<td>US Patent US7718389 suggested ascorbate can increase coelenterazine stability.</td>
		</tr>
		<tr>
			<td>Na2SO3</td>
			<td>Sigma</td>
			<td>S0505-250G</td>
			<td>US Patent US8367357 suggested sulfite may decrease background due to BSA</td>
		</tr>
		<tr>
			<td>Coelenterazine (native)</td>
			<td>Nanolight / Prolume</td>
			<td>3035MG</td>
			<td>Stock solution: 1 mg/ml in acidified MeOH (2.36 mM)</td>
		</tr>
		<tr>
			<td>OptiPlate-96, White Opaque 96-well Microplate</td>
			<td>Perkin Elmer</td>
			<td>6005290</td>
			<td>Any opaque white 96 well plate should be sufficient. Clear bottom plates will also work, however some signal will be lost.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy H1 Hybrid plate reader or equivalent</td>
			<td>BioTek</td>
			<td>8041000</td>
			<td>A plate reader with luminescence detection and 96-well plate capabilities is required.</td>
		</tr>
		<tr>
			<td>8-channel VOYAGER Pipette (50-1250 &#181;L)</td>
			<td>Integra</td>
			<td>4724</td>
			<td>An automated multichannel pipette is extremely useful for rapid addition of luciferase reagents and plating cells in 96 well format</td>
		</tr>
		<tr>
			<td>8-channel 200 &#181;L pipette</td>
			<td>Transferpette S 20-200 &#181;L</td>
			<td>2703710</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring relative insulin secretion using a co-secreted luciferase surrogate

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, depending on the specific assay. Secreted luciferase assays expedite results and lower the assay cost per sample substantially. Here we present a relatively underused approach to gauge insulin secretory activity from pancreatic &#946; cells by using Gaussia luciferase genetically inserted within the C-peptide. During proteolytic processing of proinsulin, the C-peptide is excised releasing the luciferase within the insulin secretory vesicle where it is co-secreted with insulin. Results can be obtained within minutes after sample collection because of the speed of luciferase assays. A limitation of the assay is that it is a relative measurement of insulin secretion and not an absolute quantitation. However, this protocol is economical, scalable, and can be performed using most standard luminescence plate readers. Analog and digital multichannel pipettes facilitate multiple steps of the assay. Many different experimental variations can be tested simultaneously. Once a focused set of conditions are decided upon, insulin concentrations should be measured directly using antibody-based assays with standard curves to confirm the luciferase assay results.

To gauge the performance of the assay under control conditions, a simple glucose dose-response curve or a stimulation using the diazoxide paradigm can be completed. In the case of the former, pre-incubating the cells for 1 h in glucose-free conditions followed by treating for 1 h with increasing glucose concentrations should result in very little secretory activity at and below 5 mM, while increased secretion is observed above 8 mM glucose (Figure 2). Stimula...

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Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

This protocol describes how to perform rapid low-cost luciferase assays at medium-throughput using an insulin-linked Gaussia luciferase as a proxy for insulin secretion from beta cells. The assay can be performed with most luminescence plate readers and multichannel pipettes.

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, ...

This protocol describes an alternate rapid approach for assaying insulin secretion from beta cell lines to expedite drug discovery and characterization. This approach is useful because multiple treatments can be tested simultaneously, the results are available immediately following the experiment, and this assay is more affordable than others. The application of this method towards screening for new small molecule modulators of insulin secretion may identify lead compounds for future diabetes therapies.Increased knowledge of beta cell function can contribute to greater understanding of protein or neurotransmitter secretion in general. This insulin secretion reporter has been established to work in a lentiviral system in other beta cells lines and in human islets. This assay should be very straightforward to individuals familiar with cell culture and operating multichannel pipettes.Before planning any experiments, verify that your parental or stable beta cell lines are responding properly to glucose using an insulin ELISA. Begin by preparing Krebs-Ringer Bicarbonate Buffer or KRBH and gaussia luciferase working solution according to the manuscript directions. Grow MIN6 cells in T75 flasks using standard cell culture techniques.Prepare the InsGLuc MIN6 cells for plating by washing a confluent flask of cells twice with PBS and adding two milliliters of trypsin. Incubate the cells at 37 degrees celsius for about five minutes, or until the cells dissociate from the flask. Determine the cell concentration and dilute the cells in complete media to one million cells per milliliter, which equates to 100, 000 cells per 100 microliters in each well of a 96 well plate.The cells should be sufficiently confluent for the assay after three to four days. On the day of the assay, prepare enough glucose free KRBH for the experiment and set up a reservoir. Decant the medium from the 96 well plate by quickly inverting it over a laboratory sink and then blotting firmly on a stack of paper towels to remove excess medium.Wash the plate twice with 100 microliters of glucose free KRBH per well, and if performing drug treatments, add drugs to the KRBH according to the manuscript directions. Then, add 100 microliters of glucose free KRBH to each well and incubate at 37 degrees celsius for one hour. After the incubation, decant the buffer and blot the plate on a paper towel.Add 100 microliters of glucose free KRBH per well to wash away any accumulated background and then decant the plate. Add control and stimulatory conditions with or without drug treatments at 100 microliters per well and incubate the plate at 37 degrees celsius for one hour. It's important to include basal and stimulated controls on each plate in order to determine the responsiveness of the cells in a given experiment.If the cells have lost their response over time, reculture them from a fresh liquid nitrogen stock. Carefully collect 50 microliters of supernatant using a multichannel pipette, transfer it to an opaque white 96 well assay plate, and keep the sample at room temperature on the bench until ready for the luciferase assay. Prepare the gaussia luciferase assay working solution by pipetting the required amount of CTZ solution into the GLuc assay buffer, taking care to prevent the CTZ from warming.Use a multichannel pipette to quickly add the GLuc assay working solution to the plate with the KRBH supernatants. If there are any droplets on the sides of the wells, briefly spin the plate in a tabletop, swing bucket centrifuge. Then put the plate in the plate reader and read the luminescence with a 0.1 second integration time.This assay has been tested under control conditions by measuring insulin secretion as a response to increasing glucose concentrations. Very little secretory activity is observed below five millimolar glucose, and increased secretion is observed above eight millimolar. The cells exhibit the expected secretory response when challenged with the diazoxide paradigm.Diazoxide treatment blocks insulin secretion if there is no extracellular potassium chloride. When potassium chloride is present, secretion can be amplified with further addition of glucose. It has also been demonstrated that the InsGLuc MIN6 cells respond as expected to secretion modulating compounds such as potassium chloride, diazoxide, PMA, and epinephrin.Following this procedure, critical results should be confirmed by analyzing KRBH supernatants in antibody based ELISA or HTRF assays which allow for the determination of insulin concentrations. A lot of pioneering work has been done by a group at the Broad Institute, which paved the way for me to increasingly use this assay in my research. Multiple research groups including us and the group at the Broad, are using this approach in drug discovery efforts in the field of diabetes.

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Research

JoVE Journal

Biology

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

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 ...

Measuring Blood Pressure in Mice using Volume Pressure Recording, a Tail-cuff Method

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume. A specially designed differential pressure transducer and an occlusion tail-cuff measure the total blood volume in the tail without the need to obtain the individual pulse signal. Special attention is afforded to the length of the occlusion cuff in order to derive the most accurate blood pressure readings. VPR can easily obtain readings on dark-skinned rodents, such as C57BL6 mice and is MRI compatible. The CODA system provides you with measurements of six (6) different blood pressure parameters; systolic and diastolic blood pressure, heart rate, mean blood pressure, tail blood flow, and tail blood volume. Measurements can be made on either awake or anesthetized mice or rats. The CODA system includes a controller, laptop computer, software, cuffs, animal holders, infrared warming pads, and an infrared thermometer. There are seven different holder sizes for mice as small as 8 grams to rats as large as 900 grams.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. This tail-cuff system uses Volume Pressure Recording (VPR) to measure the blood pressure by determining the tail blood volume.

The CODA 8-Channel High Throughput Non-Invasive Blood Pressure system measures the blood pressure in up to 8 mice or rats simultaneously. The CODA ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

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 ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.