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 solution containing 5 mM KCl, 120 mM NaCl, 15 mM HEPES (pH 7.4), 24 mM NaHCO3, 1 mM MgCl2, 2 mM CaCl2, and 1 mg/mL radioimmunoassay-grade bovine serum albumin (BSA). Glucose is to be added where specified from a 2 M stock. 
	NOTE: KRBH is used to incubate the cells with and without stimulation in order to assess insulin and/or Gaussia luciferase secretion.</li>
	<li>Prepare coelenterazine (CTZ) stock solution as follows. Prepare acidified methanol by adding 106 &#181;L of concentrated HCl to 10 mL of methanol. Next, dissolve lyophilized CTZ in acidified methanol at 1 mg/mL and store at -80 &#176;C in screw-cap tubes. 
	NOTE: These stocks retain sufficient activity in routine luciferase assays, even after 1 year of proper storage.</li>
	<li>Prepare Gaussia luciferase (GLuc) assay buffer based upon the literature9 as well as patent information10 to aid in half-life of the Gaussia luciferase assay in 96 well plate format. Use the formula: 25 mM Tris pH 8, 1 mM EDTA, 5% glycerol, 1 mg/mL Na2PO4, 300 mM sodium ascorbate, 200 mM Na2SO3 in water. Freeze stocks at -20 &#176;C. After thawing, store the buffer at 4 &#176;C.</li>
	<li>To prepare Gaussia luciferase working solution, add 4.2 &#181;L/mL of the 1 mg/mL (2.36 mM) CTZ stock solution to GLuc assay buffer. This results in a 2x working solution of 10 &#181;M CTZ which will have a 5 &#181;M final concentration in the assay.</li>
</ol>
2. Culture of InsGLuc MIN6 cells and seeding for secretion assays
<ol>
	<li>To culture MIN6 cells, trypsinize and seed the cells once per week using standard cell culture techniques. Change media on the cells every two to three days. Include appropriate selection antibiotic, such as 250 &#181;g/mL of G418 in the media.
	<ol>
		<li>To provide a sufficient number of cells weekly for experiments, maintain the cells in T75 flasks. Seeding 6 x 106 cells per T75 in 10 mL of media will typically yield 30 x 106 to 40 x 106 cells total per T75 after 7 days of culture.</li>
	</ol>
	</li>
	<li>To prepare cells for plating into 96-well plates, wash a confluent T75 of InsGLuc MIN6 cells twice with PBS and add 2 mL of trypsin. Incubate at 37 &#176;C for ~5 min or until the cells dissociate from the flask. Determine the cell concentration per milliliter.
	<ol>
		<li>Dilute the cells in complete media to 1 x 106 cells/mL to result in 1 x 105 cells in 100 &#181;L per well in a 96-well plate. The cells should be sufficiently confluent for the assay after 3&#8211;4 days. 
		NOTE: To extend the culture period, half the cell concentration can be plated. Media changes are not required prior to the day of the assay unless the cells are to be subjected to experimental treatments.</li>
	</ol>
	</li>
</ol>
3. Glucose-stimulated Gaussia luciferase secretion assay
<ol>
	<li>On the day of the assay prepare enough KRBH for the experiment (step 1.2). A minimum of 50 mL of KRBH per 96-well plate is required, therefore prepare at least 75 mL to ensure sufficient buffer for the experiment. Prepare extra buffer if different combinations of drug treatment conditions will require KRBH for dilution.</li>
	<li>Prepare a reservoir with glucose-free KRBH. Decant the medium from 96-well plate(s) by quickly inverting the plate over a laboratory sink and then blot firmly on a stack of paper towels to remove excess medium.</li>
	<li>Using either an electronic or manual 8-channel pipette, pipette 100 &#181;L /well KRBH from the reservoir across the 96-well plate(s). Repeat for a total of two washes.</li>
	<li>(Optional step of acute compound treatments) If not performing drug treatments, proceed with steps 3.5 and 3.6 without modification. To test the effects of small molecules on insulin secretion, compounds can be added to the cells during the preincubation period, stimulation period, or both.
	<ol>
		<li>One technique is to add compounds in batch to the KRBH in 1.5 mL tubes and use an adjustable 8-channel digital pipette to transfer the drug-KRBH from the tubes to cells in 96-well plates. 
		NOTE: If an adjustable pipette is not available, a replica 96-well plate of drug-KRBH can be made and a standard 8-channel pipette can be used to transfer buffer. Further modifications to the treatment paradigm can be made to treat cells for 24 h in media prior to the assay, as previously described1,8.</li>
	</ol>
	</li>
	<li>Add 100 &#181;L of KRBH containing the desired concentration of glucose or compounds and place the dish in the 37 &#176;C incubator for 1 h. 
	NOTE: Depending on the experimental layout, it is extremely helpful to have an electronic multichannel pipette that allows transitioning the channel distances from a column of eight 1.5-mL tubes to the 8 rows of a 96-well plate.</li>
	<li>After the 1 h of preincubation, decant the buffer into the sink and blot firmly on paper towel. Add 100 &#181;L of glucose-free KRBH per well to wash away accumulated background of Gaussia luciferase. Decant the plate again and add control and stimulatory conditions to the plate at 100 &#181;L per well. Place the dish in the 37&#176;C incubator for 1 h.</li>
	<li>Carefully collect 50 &#181;L of supernatant using a multichannel pipette, changing tips between treatment conditions as necessary, and transfer the supernatant to a clean opaque white 96-well assay plate. 
	NOTE: White-walled clear-bottom plates can be used if necessary, although a significant amount of luciferase signal will be lost.</li>
	<li>After collection of 50 &#181;L of KRBH supernatant, the sample can be assayed immediately. If necessary, seal and store samples at 4 &#176;C for a few days (GLuc activity half-life ~6 days) or -20 &#176;C for up to one month11,12.</li>
</ol>
4. Secreted Gaussia luciferase assay
<ol>
	<li>To prepare the GLuc assay working solution, pipette the required amount of CTZ stock solution (4.2 &#181;L/mL) into GLuc assay buffer. To prevent warming the CTZ, pipette the CTZ quickly at the -80 &#176;C freezer or keep the tube on dry ice.</li>
	<li>Using an electronic multichannel pipette, quickly add 50 &#181;L of the GLuc assay working solution per well across the 96-well dish containing the collected KRBH supernatants. If there are any droplets on the sides of any wells, briefly spin the plate in a table-top swing-bucket centrifuge.</li>
	<li>Read the luminescence in a suitable plate reader within a few minutes and read each well with a 0.1 s integration time.</li>
</ol>

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<li>Mourad, N. I., Nenquin, M., Henquin, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amplification+of+insulin+secretion+by+acetylcholine+or+phorbol+ester+is+independent+of+beta-cell+microfilaments+and+distinct+from+metabolic+amplification%5BTitle%5D)+AND+%22Molecular+%26+Cellular+Endocrinology%22%5BJournal%5D)">Amplification of insulin secretion by acetylcholine or phorbol ester is independent of beta-cell microfilaments and distinct from metabolic amplification.</a> Molecular & Cellular Endocrinology. 367 (1-2), 11-20 (2013).</li>
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<li>Cheng, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+passage+MIN6+cells+have+impaired+insulin+secretion+with+impaired+glucose+and+lipid+oxidation%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">High passage MIN6 cells have impaired insulin secretion with impaired glucose and lipid oxidation.</a> PLoS One. 7 (7), e40868(2012).</li>
<li>Head, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Connexin-36+Gap+Junctions+Regulate+In+Vivo+First-+and+Second-Phase+Insulin+Secretion+Dynamics+and+Glucose+Tolerance+in+the+Conscious%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">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/pubmed?term=((Gap+junctions+and+other+mechanisms+of+cell-cell+communication+regulate+basal+insulin+secretion+in+the+pancreatic+islet%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">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>
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The authors have nothing to disclose.

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>&lt;strong&gt;Cell culture materials&lt;/strong&gt;</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>&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;Secretion assay reagents&lt;/strong&gt;</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>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Thermo-Fisher Scientific</td><td>15568414</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Thermo-Fisher Scientific</td><td>M9272-500G</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C-7902</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;Optional drugs for stimulation experiments&lt;/strong&gt;</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 &amp;micro;M in DMSO</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;Guassia assay materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Disodium phosphate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</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>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;3&lt;/sub&gt;</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>&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>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 &amp;micro;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 &amp;micro;L pipette</td><td>Transferpette S 20-200 &amp;micro;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...

Watch this Scientific Journal Video about Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate at JoVE.com

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, ...

measuring-relative-insulin-secretion-using-co-secreted-luciferase

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Biology

7.4K Views.  University of Texas Southwestern Medical Center. 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.

Video: Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

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.

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 ...

Genetics

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

Biochemistry

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 ...

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.

Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method&#x2014;adapted from a coculture model of synaptogenesis&#x2014;for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

Medicine

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Intracellular Signaling Events and Hormone Secretion in Human Pseudoislets

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

Author Spotlight: Investigating Islet Abnormalities and Function with a Pseudoislet Protocol 

This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system.

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, ...

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

Summary

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