Respirometry ad alta risoluzione per misurare la funzione mitocondriale di cellule Beta intatte in presenza di composti naturali

The overall goal of this approach is to directly assess the respiratory function of pancreatic beta cells, under insulin stimulatory conditions. This can also be used to observe the effect of different compounds on beta cell respiratory function. This method can help answer key questions in the pancreatic beta cell physiology field, such as whether a certain compound impacts pancreatic beta cell function via changes in respiration.

The main advantage of this technique, is that it can be used to directly assess respiratory function in pancreatic beta cells, under basal and insulin stimulatory conditions. Visual demonstration of this method is critical as the cell preparation and machine usage steps are difficult to learn, due to the required precision in cell preparation and measurement of respiration. To begin, culture INS-1-derived 832/13 beta cells in supplemented RPMI 1640.

Remove the cells from a T75 flask, by adding two milliliters of 0.25%trypsin, and incubating at 37 degrees Celsius for 10 minutes. Then, neutralize the trypsin by adding eight milliliters of complete RPMI 1640 media. Dilute 100 microliters of the cell volume in 900 microliters of PBS, and count the cells using a hemocytometer.

Then, plate the cells at a density of one million cells per milliliter, in a 10 centimeter dish. Culture the cells in a humidified incubator at 37 degrees Celsius and 5%CO2, before beginning respiration experiments. Change the media 24 hours after plating and culturing the 832/13 cells in a humidified incubator at 37 degrees Celsius and 5%CO2.

Then, treat the cells with vehicle control or cocoa derived, epicatechin monomer, for a final concentration of 100 nanomolar. Follow addition of treatment with culture for an additional 24 hours in a humidified incubator, at 37 degrees Celsius and 5%CO2. Then, wash the cells in 1x low glucose secretion assay buffer, or SAB for five minutes.

Aspirate the buffer and incubate the cells in 1x low glucose SAB for three hours, changing the buffer every hour. Following incubation, remove the cells from the dish with one milliliter of 0.25%trypsin. Combine the cells in trypsin from the dish treated with vehicle control, with four milliliters of SAB in a 15 milliliter conical tube.

Similarly, combine the dish treated with the compound of interest, with a buffer. Dilute 100 microliters of the cell volume in 900 microliters of PBS, and count the cells using a hemocytometer. Dilute 100 microliters of the cell volume in 900 microliters of PBS, and count the cells using a hemocytometer.

The data demonstrates, that a concentration of one million cells per milliliter is the most effective. After preparing the high-resolution respirometer as detailed in the text protocol, add 2.4 milliliters of 1x low glucose SAB buffer to each oxygraph chamber. Stir the buffer continuously using magnetic stir bars in the chamber at 750 rpm and 37 degrees Celsius, with a data recording interval of 2.0 seconds.

To do so, select the F7 button, and open the tab labeled, systems. Push plungers all the way in and then retract them to the wrench aeration setting. Let the machine equilibrate for a minimum of one hour until stable oxygen flux is obtained.

Next, set the Machine at 37 degrees Celsius for the duration of the experiment. Push the F7 button and open the tab labeled, Oxygen O2, to set the polarization voltage to 800 millivolts with a gain of two. Equilibrate the oxygen concentration of the SAB buffer for at least 30 minutes, until the change in oxygen concentration is stable.

Then, select a region where the change of oxygen concentration is stable to establish background measurement of change in oxygen concentration, by pushing the shift key, left clicking on the mouse and dragging the mouse across the selected region. Click on the letter associated with the selected region and change it to R1 for each trace corresponding with each of the two chambers. Double-click on the O2 calibration box in the bottom left and right corners of the screen.

Click the Select Mark button for the air calibration as R1, and then select calibrate and copy to clipboard for both chambers. Next, load 2.4 milliliters of sample in each chamber, loading one chamber with the control vehicle treated cells and one chamber with compound treated cells in 1x low glucose SAB. Push the plunger in all the way and aspirate the residual volume.

Stir the cells continuously throughout the experiment at 750 rpm and 37 degrees Celsius for all subsequent steps. Make a mark by clicking F4 and labeling the mark as cells when samples are loaded. Measure the samples for 30 minutes.

After signal stabilization, select a region of the change in oxygen concentration corresponding to the low glucose conditions. Then, add 12.5 microliters of a 45%sterile glucose solution into each chamber through the titanium loading port using a syringe. Make a mark labeled glucose, when the treatment is added.

Let the signal stabilize and record cellular respiration until a stable oxygen flux is achieved. At this point, select the region of the change in oxygen concentration to represent the 16.7 millimolar glucose reading, which corresponds with stimulatory conditions. After signal stabilization is reached, add one microliter of five millimolar Oligomycin A into each chamber through the loading port.

Make a mark labeled, OligoA, when the treatment is added. Let the signal stabilize and record cellular respiration until a stable oxygen flux is achieved. Once stable oxygen flux is achieved, select this area of the curve.

Oligomycin A inhibits ATP synthase and thus the only oxygen flux occurring is via leak of electrons and not oxidative phosphorylation. Next, add one millimolar FCCP in one microliter increments until a maximum respiration rate is established. This represents maximal uncoupled respiration.

Between three and four microliters of FCCP, is sufficient to induce maximal uncoupled respiration of INS-1-derived 832/13 beta cells. Make a mark labeled, FCCP when the treatment is added. Let the signal stabilize and record cellular respiration until a stable oxygen flux is achieved.

Then, select this area of the curve. FCCP is an uncoupling agent, allowing measurement of uncoupled respiration. After signal stabilization is reached, add one microliter of five millimolar Antimycin A into each chamber through the loading port.

Make a mark labeled, AntiA, when treatment is added and repeat the curve selection procedure. Enter the number of cells per milliliter used in the assay, by pushing the F3 button of the respirometer analysis program. Change the units to cells per milliliter, enter the cellular concentration and change the medium to SAB.

Select the background readings to normalize the data by selecting and entering into the oxygen calibration form. Make selections of readings from 2.5 millimolar glucose, 16.7 millimolar glucose, Oligomycin A, FCCP, and Antimycin A.Within the respirometer analysis program, enter the protein concentration and select the appropriate average values for each treatment during the respiration measurement. Then, click F2 and click the copy to clipboard function.

This exports the data for use in other analysis programs. Use the O2 slope negative values for calculations. Compile the data for three to five independent runs of vehicle treated controls and natural compounds treated cells to determine the effect on intact cellular respiration of INS-1-derived 832/13 beta cells.

Intact INS-1-derived 832/13 beta cells demonstrate a glucose induced increase in oxygen utilization. It is critical that the appropriate number of cells are used. The results gathered in this experiment demonstrate that one million cells per milliliter is the optimal amount.

INS-1-derived 832/13 beta cells treated with curcumin show no change in overall respiration. Conversely, INS-1-derived 832/13 beta cells treated with monomeric cocoa epicatechins had increased respiration at 2.5 millimolar glucose and 16.7 millimolar glucose. Increased respiration is also observed during the leak state which is the basal, non-phosphorylating respiratory state, as well as the electron transport system respiration or the ETS state, which is maximal respiration.

After watching this video, you should have a good understanding of how to directly assess the respiratory function of pancreatic beta cells under insulin stimulatory conditions. Once mastered, this technique can be done in five hours if it is performed properly. While attempting this procedure, it's important to remember to pretreat the cells with low glucose SAB buffer, and get accurate cell counts for the assay.

Generally, individuals new to this method will struggle to get the right amount of cells for effective measurements. With too many cells, the signal is difficult to evaluate and with too few, the signal isn't high enough to consistently measure. The implications of this technique extend toward therapy or diagnosis of diabetes and any condition impacted by changes in pancreatic beta cell function, because mitochondrial respiration is a critical component of how insulin secretion works.

Following this procedure, other methods like glucose-stimulated insulin secretion can be performed in order to answer additional questions, like, what is the effect of these compounds on insulin secretion? Though this method can provide insight into beta cell function, it can also be applied to other systems, such as muscle, liver, adipose and other mitochondrial respiratory analyses.