The authors have nothing to disclose.

1. INS-1E Cells: Maintenance and Plating
<ol>
	<li>Maintain INS-1E cells in a humidified 37 &#730;C/5% CO2 incubator and cultured with RPMI 1640 medium supplemented with 5% (v/v) heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 U/mL Penicillin/Streptomycin solution, 50 &#181;M &#946;-mercaptoethanol. Culture cells in 10 mL complete RPMI 1640 medium (per plate), until they reach 80 - 90% confluence, when they can be trypsinized and passaged or used for the insulin secretion assay.</li>
	<li>Day 1: Aspirate media and wash cells once with 5 mL of pre-warmed PBS. Add 0.5 mL of trypsin (0.025%) diluted 1:1 in 0.5 mL PBS to trypsinize the cells and incubate for 3 - 4 min at 37 &#730;C. Deactivate trypsin by adding 9 mL complete media and transfer cells to a 15 mL centrifuge tube by pipetting.</li>
	<li>Pellet cells by centrifugation and re-suspend the cell pellet in approximately 5 mL fresh media.</li>
	<li>Take 10 &#181;L of the re-suspended cells and mix with 10 &#181;L Trypan Blue vital dye to check for cell viability. Count live and dead cells in 10 &#181;L of this mixture using a hemocytometer. Viability level should be above 90%. Dilute the re-suspended cells in fresh media to 1 million cells per mL.</li>
	<li>Seed 0.5 mL INS-1E cells per well in a poly-L-Lysine pre-coated 24-well plate, at a density of 500,000 cells/well.</li>
	<li>Day 2: Remove media 18 - 24 h after plating and add 500 &#181;L/well of fresh RPMI 1640 media. Incubate the cells for another 24 h to allow cells to fully recover from the prior passaging step. This additional time permits the cells to spread on the tissue culture plate.</li>
</ol>
2. Insulin Secretion Assay (Day 3)
<ol>
	<li>Prepare KRB buffer: 132.2 mM NaCl, 3.6 mM KCl, 5 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 1.5 mM CaCl2,&#160;and 0.001 g/mL bovine serum albumin (BSA), pH 7.4.</li>
	<li>Aspirate media from cells and wash twice with pre-warmed PBS.</li>
	<li>For the glucose starvation step, add 450 &#181;L/well KRB (containing BSA) without glucose for 1 h at 37 &#730;C/5% CO2.</li>
	<li>During the glucose starvation step, prepare serial dilutions of the drug(s) in KRB containing 200 mM Glucose (10x concentration).
	<ol>
		<li>Prepare drug at 10x the final concentration in KRB supplemented with 200 mM glucose (also 10x the final assay glucose concentration). If the drug stock (plus the added 10x glucose) is in DMSO, make sure DMSO percentage is kept consistent throughout the assay (ideally final percentage of less than 0.1% DMSO).</li>
		<li>For dopamine and quinpirole treatments, use a final assay drug concentration range of 100 &#181;M to 100 pM (from highest to lowest concentration), with the last point of the dose response containing no drug. For bromocriptine, use a final assay concentration range of 10 &#181;M to 10 pM, with the last point of the dose response being the drug-free control.</li>
	</ol>
	</li>
	<li>After glucose starvation, add drug serial dilutions to the assay to produce a dose response.</li>
	<li>Add 50 &#181;L/well of each serial dilution to the corresponding wells (total assay volume 500 &#181;L).</li>
	<li>For the glucose stimulation step, incubate cells with the respective drug serial dilutions (in the presence of 20 mM glucose) for 90 min at 37 &#730;C/5% CO2. Include a set of control wells: (1) stimulation with 20 mM glucose alone in the absence of any additional drug and (2) cells that are neither stimulated with drug nor glucose (which provides a basal rate of secretion).</li>
	<li>After the stimulation step, carefully remove the supernatants (use directly or store at 4 &#730;C). 
	NOTE: An additional 5 min gentle centrifugation step (600 x g, 1 min) may be introduced at this point to remove any remaining cells in the assay supernatants.</li>
</ol>
3. HTRF to Measure Insulin Secretion
<ol>
	<li>Dilute assay supernatants 1:10 in KRB (without BSA), preferably in clear 96-well plates.</li>
	<li>Prepare the insulin standard curve for the HTRF insulin assay (Table 1).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Standard stock solution 500 ng/ml</td>
			<td>Serial dilutions</td>
			<td>Working [insulin] ng/ml</td>
		</tr>
		<tr>
			<td>STD 7</td>
			<td>30 &#181;l Stock + 140 &#181;l KRB</td>
			<td>150</td>
		</tr>
		<tr>
			<td>STD 6</td>
			<td>30 &#181;l STD 7 + 45 &#181;l KRB</td>
			<td>60</td>
		</tr>
		<tr>
			<td>STD 5</td>
			<td>30 &#181;l STD 6 + 45 &#181;l KRB</td>
			<td>24</td>
		</tr>
		<tr>
			<td>STD 4</td>
			<td>30 &#181;l STD 5 + 45 &#181;l KRB</td>
			<td>9.6</td>
		</tr>
		<tr>
			<td>STD 3</td>
			<td>30 &#181;l STD 4 + 45 &#181;l KRB</td>
			<td>3.84</td>
		</tr>
		<tr>
			<td>STD 2</td>
			<td>30 &#181;l STD 3 + 45 &#181;l KRB</td>
			<td>1.54</td>
		</tr>
		<tr>
			<td>STD 1</td>
			<td>30 &#181;l STD 2 + 45 &#181;l KRB</td>
			<td>0.61</td>
		</tr>
		<tr>
			<td>STD 0</td>
			<td>45 &#181;l KRB</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="3">Note: STD Stock is 500 ng/ml</td>
		</tr>
	</tbody>
</table>
Table 1. Serial dilutions to make the insulin standard curve.
<ol start="3">
	<li>Add the standard curve samples and the diluted assay supernatants to the HTRF plate. The measurement of secreted insulin by HTRF can be carried out in either a 96-well or a 384-well plate format, keeping in mind that the assay volume has to be re-adjusted. Use 10 &#181;L/well sample in 96-well white half-area plates or 5 &#181;L/well in a 384-well white low-volume, round-bottom plate (see Table of Materials).</li>
	<li>Prepare antibody mix in detection buffer (see Table of Materials) in a 1:2 donor (cryptate)/acceptor (XL-665) ratio. 
	NOTE: Further specific details about the HTRF assay are available from the manufacturer.</li>
	<li>Add antibody mix to the assay at 30 &#181;L/well (for a 96 well-plate assay format) or 15 &#181;L/well (for a 384-well plate assay format).</li>
	<li>Seal the plate and incubate at room temperature.</li>
	<li>Read plate after 2 h, 4 h, and/or overnight incubation with antibodies using the plate reader and the appropriate HTRF optic module (337 665 620 nm) (see Table of Materials and manufacturer&#39;s instructions). Set the integration start at 60 &#181;s and the integration time at 400 &#181;s. Use 200 flashes per well. 
	NOTE: These parameters were based on the use of our particular reader. The readout at 620 nm and 665 nm may vary between different instruments. This is one of the reasons we suggest using the 665/620 ratio. In calculating this ratio, any potential differences from reader to reader will be normalized and provide consistent values irrespective of the instrument used to measure HTRF.</li>
</ol>
4. Data Analysis and Normalization
<ol>
	<li>Calculate the insulin concentrations of the assay wells via extrapolation of ratiometric fluorescence readings (665 nm/620 nm) to a second order quadratic polynomial curve (Figure 2).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57531/57531fig2.jpg" /> 
Figure 2: Insulin standard curve. Human insulin stock of known concentrations was used to generate the insulin standard curve. The resulting HTRF ratios (665 nm/ 620 nm) were plotted against the insulin concentrations. The data was best fit to a second order quadratic polynomial curve (R2 = 0.99996). This is a representative standard curve. Error bars = SEM. <a href="/files/ftp_upload/57531/57531fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>With the extrapolated data as ng/mL of insulin secreted, normalize (insulin secreted in response to increasing ligand concentrations) to the average value of the % maximum insulin secretion assay wells (20 mM glucose alone condition).</li>
	<li>Use the curve fit (R2) from a single experiment to calculate the intraplate variation. Within the experiment, derived the individual R2 values from the intra-experimental duplicates, allowing calculation the standard error of the mean for the curve fit.</li>
	<li>For determining the interplate variation, use the data from at least three individual experiments to calculate the standard error of the mean for the R2 value of the collective curve.</li>
</ol>

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We thank Nicolas Pierre (Cisbio Bioassays) for helpful advice and Dr. Pierre Maechler (University of Geneva) for generously providing INS-1E cells. This work was supported by funding from the Department of Defense (grant PR141292 to Z.F.), and the John F. and Nancy A. Emmerling Fund of The Pittsburgh Foundation (to Z.F.).

The HTRF insulin assay described here offers a rapid, efficient system to measure insulin secretion from a cultured cell-based system. Among its most important advantages, this assay offers a low background signal due to the high signal-to-noise ratio. Additionally, we have confirmed that the HTRF signal is stable for extended periods of time (&#62;24 h)7. Nevertheless, since the insulin-binding monoclonal antibodies quickly reach binding saturation after addition to the assay, kinetics of antibod...

The regulation of energy metabolism is fine-tuned by a major anabolic hormone, insulin. Insulin is synthesized and released by pancreatic beta cells in response to increased extracellular glucose levels. The released insulin triggers the uptake of glucose by insulin-sensitive tissues1,2. Physiologically, this is linked to the elevation of glucose concentration after a meal, followed by secretion of insulin to regulate glucose uptake. Disturbances in glucose homeostasis lead to metabolic impairments culminating in insulin resistance and ultimately in the onset of type 2 diabetes2,3,4.
Although insulin secretion has been extensively studied, its regulatory mechanisms remain poorly understood. A critical area of investigation has been identification of novel modulators of insulin secretion by beta cells5,6,7,8. These studies require a better understanding of the coupling relationship between glucose stimulation and insulin secretion. Therefore, the ability to accurately monitor and quantify the levels of glucose-stimulated insulin secretion (GSIS) has been essential. To date, however, only a limited number of methods were available to allow quantification of GSIS using insulin-secreting cell lines and/or pancreatic islets. One is radioimmunoassay (RIA), which utilizes radioisotope-tagged insulin and antibodies. The main limitations of this approach include safety issues due to the handling and disposal of radioactive materials. Additionally, this method is labor-intensive, involving multiple long washing and incubation steps. Enzyme-linked immunosorbent assay (ELISA) is another costly and labor-intensive approach that utilizes antibodies for insulin detection. Variation in antibody affinities and in the efficiency of recognizing insulin are limiting factors of this method and can affect the reproducibility of the results. Neither ELISA nor RIA was designed for high-throughput experiments. AlphaScreen is a homogeneous assay used for detecting and measuring levels of insulin secretion. AlphaScreen technology is based on the conversion of ambient oxygen into an excited oxygen singlet state that can react with chemiluminescent species, resulting in the generation of chemiluminescence. Because the assay is homogenous, many of the washing steps associated with RIA and ELISA are eliminated. However, the instability of the signal due to the nature of the reaction is a limiting factor that may affect the readout of the assay. (TR-PINCER, developed by Heyduk and colleagues9, is another homogenous approach to insulin measurement based on the binding of two separate antibodies to different epitopes on the insulin molecule. The antibodies are each chemically linked to double stranded DNA with short complementary single stranded DNA overhangs. Binding of the antibodies to insulin brings them together and leads to a double stranded DNA duplex. Each antibody is also associated with a respective donor or acceptor fluorophore, and the association of the DNA duplex brings together these fluorophores to generate F&#246;rster resonance energy transfer (FRET). One potential limitation of TR-PINCER, however, rests with the FRET itself. The inability to rapidly dissipate background fluorescence during the FRET reaction may lead to relatively high levels of background fluorescence and a low signal to noise ratio within the assay. Therefore, a need still exists for a reliable, robust, and cost-effective assay for quantifying GSIS in a high-throughput manner.
Recent advances in biophysics have culminated in the development of a homogeneous time-resolved fluorescence energy resonance transfer (HTRF) based assay. Specifically, while the energy transfer within the assay may be described as FRET-based, more accurately, HTRF relies on luminescence energy resonance transfer (LRET)10 which is the non-radiative transfer of energy between the donor and acceptor species11,12,13. This distinction is important, since the timing of a fluorescence or quenching based FRET interaction is much different than it is for LRET, though the same types of gating can be used for FRET and LRET. Moreover, the use of rare earth lanthanide cryptate compounds such as europium or terbium cryptate in HTRF produces long fluorescence half-lives12,14. This offers the unique advantage of the introduction of a time delay (&#181;sec) between donor excitation and the measurement of emission from the acceptor (i.e., time-resolved assay). This time delay allows for sufficient time for the background fluorescence to dissipate prior to measurement of acceptor emission fluorescence. Consequently, the readout is free of non-specific fluorescence and thus, a high signal-to-noise ratio is achieved (Figure 1). Furthermore, the homogenous nature of HTRF eliminates the need for washing steps to wash off the unbound species, making the assay much more rapid than ELISA or RIA-based methods.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57531/57531fig1.jpg" /> 
Figure 1: Schematic of the mechanism for HTRF insulin detection. Two independently generated monoclonal antibodies specifically recognize and bind to insulin at separate sites. These antibodies are conjugated to either the europium cryptate donor or the XL665 acceptor. Excitation of the donor at 337 nm results in emission at 620 nm. The resulting energy transfer causes XL665 to emit at a longer wavelength, 665 nm. <a href="/files/ftp_upload/57531/57531fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Here, we provide a detailed protocol for using an HTRF-based approach to determine the levels of GSIS from INS-1E cells, a well-established insulin-secreting beta cell-derived rat insulinoma cell line15. In addition, this assay may be used for identifying the pharmacological profile of molecular regulators of insulin secretion. We apply this HTRF-based insulin assay to examine dopamine D2-like receptor regulation of GSIS. Increasing studies have revealed that the neurotransmitter dopamine is an important regulator of GSIS8,16,17,18,19,20,21,22. Dopamine affects GSIS in a negative autocrine/paracrine manner via actions on the dopamine D2-like receptors (D2, D3, D4 receptors) expressed at the surface of beta pancreatic cells8,16,19. Using this assay, we confirm dopamine&#39;s role as a negative regulator of GSIS and demonstrate that the dopamine D2-like receptor agonists bromocriptine and quinpirole also reduce GSIS.

<table><tbody><tr><td>96-well white half area plate</td><td>Greiner Bio-One</td><td>82050-042</td><td></td></tr><tr><td>384-well white low-volume, round-bottom plate</td><td>Corning</td><td>15100157</td><td></td></tr><tr><td>Insulin High Range Kit</td><td>Cisbio Bioassays</td><td>62IN1PEH&amp;nbsp;</td><td>10,000 test HTRF-based insulin assay kit</td></tr><tr><td>PHERAstar FSX plate reader</td><td>BMG Labtech</td><td>FSX</td><td>Plate reader adapted for HTRF-based assay readings</td></tr><tr><td>Plate sealers</td><td>Fisher Scientific</td><td>DY992</td><td>To seal plate while antibodies incubate</td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific</td><td>3100</td><td></td></tr><tr><td>RPMI Medium 1640 (1x)</td><td>Gibco&amp;nbsp;</td><td>11875-093</td><td>[+] L-glutamine</td></tr><tr><td>Trypsin 0.05%&amp;nbsp;</td><td>Corning</td><td>25-052-CI</td><td>0.53 mM EDTA, (-) sodium bicarbonate</td></tr><tr><td>DPBS (Dulbecco&#039;s phosphate buffered saline)</td><td>Corning</td><td>21-031-CV</td><td>Without calcium and magnesium</td></tr><tr><td>Fetal Bovine Serum</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>HEPES</td><td>Gibco&amp;nbsp;</td><td>156-30-080</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco&amp;nbsp;</td><td>11360070</td><td></td></tr><tr><td>Penicillin/Streptomycin solution 100x</td><td>Corning</td><td>30-002 CT</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Sigma</td><td>M1348</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Gibco&amp;nbsp;</td><td>15250-061</td><td></td></tr><tr><td>Dopamine hydrochloride</td><td>Sigma</td><td>8502</td><td></td></tr><tr><td>Bromocriptine mesylate</td><td>Tocris</td><td>427</td><td></td></tr><tr><td>(-)-Quinpirole hydrochloride</td><td>Tocris</td><td>1061</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Calbiochem</td><td>12659</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P4832</td><td></td></tr><tr><td>Glucose</td><td>Sigma</td><td>G7021</td><td></td></tr><tr><td>DMSO (Dimethyl sulfoxide)</td><td>Sigma</td><td>276855</td><td></td></tr></tbody></table>

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.

We validated our insulin HTRF assay by generating an insulin standard curve using purified human insulin standards of predefined concentrations (Figure 2). Generation of the standard curve permitted us to extrapolate the ratiometric fluorescence readings and thus to determine the secreted insulin levels in response to the drug treatments (Figure 2). Intraplate variation for curve fitting was minimal (R2 = 0.99...

Watch this Scientific Journal Video about Homogeneous Time-resolved FÃ¶rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion at JoVE.com

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

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 ...

homogeneous-time-resolved-frster-resonance-energy-transfer-based

Research

JoVE Journal

Medicine

9.1K Views.  University of Pittsburgh. Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

Video: Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

Biochemistry

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

Bioengineering

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. 
Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved F&#246;rster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 &#181;L), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Time-resolved F&#246;rster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

Developmental Biology

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

Immunology and Infection

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 ...

Biology

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.

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, ...

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, ...

Detection of Human Islet Autoantibodies Using an Electrochemiluminescence Assay

Source: Gu, Y. et al., <a href="https://app.jove.com/v/57227">Electrochemiluminescence Assays for Human Islet Autoantibodies.</a>&#160;J. Vis. Exp. (2018)
This video showcases the electrochemiluminescence detection of islet autoantibodies in human serum. The antibodies in the serum bridge the ruthenium-conjugated antigen to the biotinylated capture antigen, forming complexes that are detected by electrochemiluminescence measurements.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Buffer Preparation

	Labeling ...

Homogeneous Time-resolved Förster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

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