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.
