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