Name: analysis of non-human primate pancreatic islet oxygen consumption
Uploaded: 2019-12-18T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption at JoVE.com

The authors would like to acknowledge the Vanderbilt High Throughput Screening Core for the use of their facilities, Agilent Biotechnologies, Dr. Paul Kievit (Oregon Health and Science University) for non-human primate islet isolations, and Eric Donahue (Vanderbilt University) for assistance with Figure 1. J.M.E. was supported by NIGMS of the National Institutes of Health under award number T32GM007347. M.G. was supported by the NIH/NIDDK (R24DK090964-06) and the Department of Veterans Affairs (BX003744).

1. Preparation of Microplate and Sensor Cartridge on the Day Prior to Running the Assay
Islets were isolated from three year old Japanese macaques as previously described13. This method is very similar to that used to isolate human islets from cadaver donors, but differs from mice, in which pancreata are often inflated with collagenase solution while the animal is under sedation and prior to organ removal. Islet retrieval was conducted in accordance with the guidelines of...

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The study of islet oxygen consumption has previously been hampered by the spherical shape of islets, their lack of adherence to culture surfaces, and the number of islets required per well. In this protocol, we highlight the efficacy of the 96-well spheroid microplate for measuring islet oxygen consumption on a small number of islets and demonstrate a technique for handling and loading islets which is technically feasible and produces consistent results.
In order for islets to adhere to the bo...

In order to maintain normal blood glucose levels, the pancreatic &#946; cell must sense elevations in glucose and secrete insulin accordingly. The coupling of insulin secretion with glucose levels is directly linked to glucose metabolism and the production of ATP through mitochondrial oxidative phosphorylation. Thus, mitochondria play a critical role in stimulus-secretion coupling1. Assessing &#946;-cell mitochondrial function can reveal defects that lead to impaired insulin secretion. The secretion of glucagon by pancreatic &#945; cells is also closely tied to mitochondrial function2. Although immortalized islet cell lines have proven useful for some types of assays, the physiology of these cells does not accurately recapitulate whole islet function, as illustrated by the potentiation of insulin secretion by glucagon3,4 and the inhibition of glucagon secretion by insulin/somatostatin5,6 in intact islets. This demonstrates the need for measuring oxygen consumption using whole, intact islets.
Techniques for the measurement of islet cell respirometry have evolved over time, from the use of oxygen-sensitive fluorescent dyes7 to solid-state sensors that directly measure oxygen consumption8. Initially designed for monolayer, adherent cells, commonly used cell culture plate systems have proven to be ineffective for pancreatic islets. As islets do not naturally adhere to the wells, they are prone to being pushed to the periphery of the culture well resulting in inaccurate measurement of oxygen consumption9. To combat this problem, specialized 24-well plates with a central depression that could contain islets were developed9. However, the 24-well plate system was limited by the large number of islets required (50-80 per well) and the number of conditions that could be tested simultaneously10. The recent development of 96-well microplates designed specifically for extracellular flux analysis in spheroids has overcome these barriers, enabling the measurement of islet respirometry with 20 or fewer islets per well10.
Here, we demonstrate the use of this system to measure oxygen consumption in islets from the Japanese macaque (Macaca fuscata), an animal model with similar islet biology to humans11,12. In this protocol, 15 macaque islets are analyzed per well. In our hands, 15 islets per well produced higher baseline oxygen consumption than fewer islets, with robust activation and repression of respiration in response to pharmacologic manipulation. We highlight the steps to prepare for the assay, an effective method for consistent loading of islets at the center of each well, and common challenges when performing this assay.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell culture dish, 60 mm X 15 mm style</td>
			<td>Corning</td>
			<td>430166</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Tak Cell and Tissue Adhesive</td>
			<td>Corning</td>
			<td>354240</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube, 50 mL</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose anhydrous</td>
			<td>Fisher Scientific</td>
			<td>BP350-1</td>
			<td>For glucose solution, 200 mg/ml, sterile filetered</td>
		</tr>
		<tr>
			<td>Disposable reservoirs (sterile), 25 ML</td>
			<td>Vistalab</td>
			<td>3054-1033</td>
			<td>for loading multichannel pipet</td>
		</tr>
		<tr>
			<td>EZFlow Sterile 0.45 &#956;m PES Syringe Filter, 13 mm</td>
			<td>Foxx Life Sciences</td>
			<td>371-3115-OEM</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td>200 mM (100x)</td>
		</tr>
		<tr>
			<td>Multichannel pipette tips</td>
			<td>ThermoFisher Scientific</td>
			<td>94410810</td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipette, 15-1250 &#956;L</td>
			<td>ThermoFisher Scientific</td>
			<td>4672100BT</td>
			<td>Recommended</td>
		</tr>
		<tr>
			<td>P20, P200, and P1000 pipettes</td>
			<td>Eppendorf</td>
			<td>2231000602</td>
			<td></td>
		</tr>
		<tr>
			<td>pH Probe</td>
			<td>Hanna Instruments</td>
			<td>HI2210-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, 20 &#956;L, 200 &#956;L, 1000 &#956;L</td>
			<td>Olympus</td>
			<td>24-404, 24-412, 24-430</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF Base Media</td>
			<td>Agilent</td>
			<td>103334-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XF Cell Mito Stress Test Kit</td>
			<td>Agilent</td>
			<td>103015-100</td>
			<td>Includes Oligomycin, FCCP, and Rotenone/Antimycin A</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Analyzer</td>
			<td>Agilent</td>
			<td>S7800B</td>
			<td>Including prep station with 37 &#176;C non-CO2 incubator</td>
		</tr>
		<tr>
			<td>Seahorse XFe96 Spheroid Fluxpak Mini</td>
			<td>Agilent</td>
			<td>102905-100</td>
			<td>Includes sensor cartridge, spheroid microplate, and calibrant</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Fisher Scientific</td>
			<td>BP328-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td>100 mM (100x)</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Olympus</td>
			<td>SZX9</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (sterile), 5 mL</td>
			<td>BD</td>
			<td>309603</td>
			<td>For sterile filtration</td>
		</tr>
		<tr>
			<td>Water (sterile)</td>
			<td>Sigma</td>
			<td>W3500-500mL</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of non-human primate pancreatic islet oxygen consumption

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate the measurement of islet oxygen consumption using a 96-well microplate designed for the measurement of oxygen consumption in spheroids. In this assay, spheroid microplates are coated with a cell and tissue adhesive on the day prior to the assay. We utilize a small volume of adhesive solution to encourage islet adherence to only the bottom of the well. On the day of the assay, 15 islets are loaded directly into the base of each well using a technique that ensures optimal positioning of islets and accurate measurement of oxygen consumption. Various aspects of mitochondrial respiration are probed pharmacologically in non-human primate islets, including ATP-dependent respiration, maximal respiration, and proton leak. This method allows for consistent, reproducible results using only a small number of islets per well. It can theoretically be applied to any cultured spheroids of similar size.

To load islets into microplate, 15 islets should be aspirated in 15 &#181;L of media, as shown in Figure 1A. Islets will naturally settle toward the bottom of the pipet tip within a few seconds. Then, the pipet tip is lowered to the bottom of the well. The tip is very slightly lifted, and a small volume (about 5 &#181;L) is pipetted out along with the islets. This technique results in consistent placement of islet at the bottom of the micropl...

Watch this Scientific Journal Video about Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption at JoVE.com

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate ...

This method allows for the quantification of oxygen consumption from small numbers of islets from relevant species, such as non-human primates and humans, allowing for assessment of mitochondrial function. This technique can be performed with small numbers of primary islets, increasing the technical replicates, allowing for the testing of multiple conditions, and improving the rigor and reproducibility of the assay. This technique assesses mitochondrial health, a major determinate of islet function and insulin secretion that is often impaired in diabetes and is a significant predictor of successful islet transplantation.It is essential to make sure that the islets are localized to the center of the wells following transfer. Failure to do so results in inconsistent oxygen consumption measurements. Demonstrating this procedure will be, Valerie Ricciardi, an undergraduate student working in my laboratory.The day before the assay, add 200 microliters of cell and tissue adhesive to 2.8 milliliters of 0.1 molar sodium bicarbonate solution and add 20 microliters of the resulting solution to the bottom of each well of a 96 well spheroid microplate. Ensure that any air bubbles are removed from the pipette tip. When all of the wells have been loaded, place the plate in a 37 degree Celsius non-carbon dioxide incubator.After one hour, aspirate the cell adhesive solution from the plate within a sterile environment and wash each well two times with 400 microliters of 37 degree Celsius sterile water. Then allow the wells to air dry for 30 to 40 minutes before covering the plate for overnight storage at four degrees Celsius. To hydrate the sensor cartridge, in a sterile environment open the sensor cartridge package and remove the contents.Place the sensor cartridge upside down next to the utility plate and fill each well of the utility plate with 200 microliters of sterile water. When all of the wells have been loaded, lower the sensor cartridge into the utility plate and place the assembled sensor cartridge and utility plate into the 37 degree Celsius non-carbon dioxide incubator overnight. On the day of the assay, remove the sensor cartridge lid and decant the water from the wells of the utility plate.Add 200 milliliters of prewarmed calibrant to each well and replace the sensor cartridge lid. Then place the sensor cartridge back into the incubator for about one hour. To prepare the islets for transfer, hand pick the islets into a 60 by 15 millimeter cell culture dish containing assembled medium to a near 100%purity.When all of the islets have been collected, load each well of the prepared spheroid microplate with 175 microliters of assembled medium and use a P20 pipette set to 15 microliters to aspirate 15 islets in 15 microliters of medium from the cell culture dish. To seed the islets onto the spheroid microplate, lower the pipette tip to the bottom of the well, barely lift the tip, and slowly dispense about five microliters of medium. To load the islets into the wells, allow the islets to settle in the pipette tip before gently releasing them directly above the bottom of the well center in a small volume of medium.Confirm that all of the islets have left the pipette tip, occasionally checking the spheroid microplate under the microscope to verify that the islets are seeded at the bottom of the plate and not stuck to the sides of the wells. When all of the islets have been transferred, place the microplate in the 37 degree Celsius non-carbon dioxide incubator. Before initiating the assay procedure, load Port A of the sensor cartridge with 20 microliters of freshly prepared 45 micromolar oligomycin, Port B with 22 microliters of freshly prepared 10 micromolar FCCP, and Port C with 25 microliters of freshly prepared 25 micromolar rotenone AA.Next, program an extracellular flux analyzer assay for a 30 minute baseline respiration period, 42 minute oligomycin measurement period, 30 minute FCCP period, and 30 minute rotenone AA period.Then run the assay following the instructions on the screen for calibrating the sensor and inserting the microplate. For islet delivery to the assay plate, 15 islets should be aspirated in 15 microliters of medium as illustrated. This technique will result in the consistent placement of islets at the bottom center of each microplate well allowing for accurate oxygen consumption measurements.In this representative experiment showing the oxygen consumption of individual wells, the wells were poorly loaded resulting in a very low baseline level of oxygen consumption and little to no response to FCCP. In this experiment, most of the wells demonstrated a significant baseline respiration and response to drugs. However, two wells exhibited no response to rotenone, suggesting that the drug was not properly released into the well.These wells were excluded from further analyses. Here, the results from a successful assay in which the wells were properly loaded with islets and correctly injected with drugs can be observed. It is beneficial to prepare the reagents and plates the day prior to performing the assay to allow the islets to be used shortly after they are isolated or received.Following the assay, the islets can be recovered for protein or DNA extraction to normalize the oxygen consumption data or to quantify the mitochondrial DNA content. The ability to measure mitochondrial function in small numbers of primary primate islets allows the testing of multiple drugs and other manipulations in a single biological replicate. Individuals working with primary primate islets should be sure to be trained in the proper and safe handling with these tissues.In particular, the use of appropriate personal protective equipment and the proper disposal of these samples.

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