The overall goal of this procedure is to induce and accurately quantify the levels of secreted insulin from insulin secreting cells. This method can help answer key questions in the diabetes field, such as the main mechanisms under glucose-stimulated insulin secretion in pancreatic beta cells. The main advantage of this technique is that it can accurately and rapidly quantify insulin secretion in response to glucose stimulation and detect potential drug effects on secretion.
The implications of this technique extend toward therapy and drug discovery for treating Type 2 diabetes, because it will allow us to better understand the mechanisms of drug-induced metabolic dysfunction. Though this method can provide insight into insulin secretion from immortalized beta cell lines, it can also be applied to other systems, such as intact pancreatic islets. Generally, individuals new to this method will struggle, because of multiple cell handling steps and drug preparations.
We first had the idea for this method when we wanted to come up with a rapid assay to investigate the actions of dopaminergic drugs on insulin secretion. Visual demonstration of this method is critical, as the cell handling steps are difficult to learn because of the delicate nature of the cells and the importance of precision to ensure consistency of the assay between experiments. First, grow the INS-1E cells as outlined in the text protocol.
Once the cells reach the desired confluency, aspirate the medium. Wash the cells with five milliliters of phosphate buffered saline. Next, add 0.5 milliliters of 025%trypsin to the cells then leave the cells in the incubator for three to four minutes at 37 degrees Celsius.
Deactivate the trypsin by adding nine milliliters of complete medium. Next, transfer the cells to a 15 milliliter centrifuge tube. Then centrifuge the cells to form a pellet.
After centrifugation, dissolve the pellet in five milliliters of fresh medium. Pipette out 10 microliters of the re-suspended cells from the tube and mix with 10 microliters of trypan blue vital dye to assess viability. Next, use a hemocytometer to count the number of living and dead cells and ensure that 90%of the cells are living.
After counting, add one million cells per milliliter in fresh RPMI 1640 medium, then seed 0.5 milliliters of 500, 000 INS-1E cells in each well of a poly-L-lysine coated 24-well plate. Leave the plate in the incubator for one whole day. The next day, remove the media and add 500 microliters of fresh RPMI 1640 medium in each of the wells.
Incubate the plate for another 24 hours for the cells to spread on the well surfaces. To perform the assay, first prepare the KRB buffer, then aspirate the medium from each of the wells and wash with pre-warmed phosphate buffered saline, twice. Next, add 450 microliters of KRB buffer, supplemented with bovine serum albumin, but no glucose, in each well.
Leave the culture plate at 37 degrees Celsius and 5%carbon dioxide for an hour. Prepare serial dilutions of the drugs in KRB buffer, supplemented with 10X final concentration of 200 millimolar glucose. After the glucose starvation step, pipette 50 microliters of serially diluted drugs in each of the corresponding wells.
For glucose stimulation, add the respective serially diluted drugs in the presence of 20 millimolar glucose. Then incubate the cells at 37 degrees Celsius for 90 minutes. Once the stimulation is over, collect the supernatants and transfer them to labeled 1.5 milliliter centrifuge tubes.
Transfer 10 microliters of each collected supernatant into a 96-well plate. Add 90 microliters of KRB to each well to achieve a one to 10 dilution ratio. Mix the dilutions to ensure homogeneity.
Then, obtain the insulin standard curve for the HTRF insulin assay. Next, add 10 microliters each of the standard insulin samples and the diluted assay supernatants to a 96-well plate. Prepare the antibody mix in a one to two donor to accept a ratio in the detection buffer.
After preparing the antibody mix, add 30 microliters of it to each of the wells in a 96-well plate. Seal the plate and incubate at room temperature. After two hours of incubation, read the plate with the antibody mix on the plate reader.
Use the appropriate HTRF optic module at 665 and 620 nanometers for the reading and use 200 flashes for each well. First, an insulin standard curve is plotted to extrapolate the actual values of insulin from the assay samples. The standard curve shows that the ratiometric fluorescence reading obtained significantly increases as a function of the pre-defined standard human insulin concentrations.
Next, the basal level of insulin secretion from the INS-1E cells is studied in the presence or absence of 20 millimolar glucose. From the plot, it shows that the cells secrete approximately 100 nanograms per milliliter of insulin in the presence of glucose. Interestingly, the insulin level is almost half at approximately 15 nanograms per milliliter in the absence of glucose.
Next, the cells are incubated in the presence of increasing concentrations of dopamine with 20 millimolar glucose. The insulin secreted is normalized to the average value of the percent maximum insulin from each well. The plot shows that dopamine inhibits GSIS, thereby indicating the functional importance of dopaminergic receptors in GSIS.
This plot shows that bromocriptine, a Type 2 diabetes drug, is more potent in inducing GSIS than dopamine. This plots shows that with increasing concentration of quinpirole, a D2 agonist, although less efficient than bromocriptine, shows similar potency like dopamine in stimulating GSIS. Once mastered, this technique can be done in four to five hours if it's performed properly.
While attempting this procedure, it's important to remember to pipette carefully and consistently. Following this procedure, other functional assays like cyclic AMP HDRF assay can be performed in order to answer additional questions, like dopaminergic drug effects on downstream signaling. After its development, this technique paved the way for researchers in the field of metabolism to explore other endocrine secretory pathways in both immortalized cell lines and pancreatic islets.
After watching this video, you should have a good understanding of how to accurately measure insulin secretion from cells using HDRF.
