Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

This method is useful in determining cellular energetics, like ATP production rate, and also to determine cellular preference towards a particular metabolite on a real time basis. To begin, add 200 microliters of XF calibrant and incubate the utility plate for at least one hour before the assay. Prepare 80 milliliters of XF assay media using supplemented XF DMEM.

Thaw oligomycin A, rotenone, and antimycin A on ice. Pipette the compounds up and down to solubilize them before use. Add three milliliters of prepared assay medium to each tube, labeled A and B.Add 26.4 microliters of 2.5 millimolar oligomycin A into tube A and 3.1 microliters of 12.67 millimolar rotenone, 4.1 microliters of 9.4 millimolar antimycin A, and 30 microliter Hook's stain into tube B.Load a 10x concentration of these inhibitors in the corresponding injection port.

And finally, 20 microliters of these compounds will be loaded into the cells in 200 microliters of assay media. Remove the cell culture microplate from the 37 degrees Celsius incubator and observe the cells under the microscope. Remove the assay medium from the water bath.

Gently wash the cells with 200 microliters of pre-warmed assay medium twice, and add 200 microliters of assay media per well. Check the cells under the microscope to ensure that the cells remain adhered to the wells. Ensure that the cells in D5 and E8 wells are adhered with a consistent monolayer and were not washed away during the washing step.

Cell imaging software uses these two wells for setting the auto focus and auto exposure. Open the desktop software in the computer next to the equipment. Check the connection status in the lower left corner of the controller software.

Go to Templates and select the XF ATP rate assay template or appropriate assay template. Select Group Definitions on the top of the screen and define the groups. select the plate map layout and assign the wells depending on the groups defined.

Verify the instrument protocol. Ensure the compounds added are correctly listed and include the project information for future references. Click on Run Assay.

This will prompt the selection of the result file storage location. Select the location to save the result file. Save the file with the assay date and click on Start Run.

Place the sensor cartridge and the utility plate on the tray and click on I'm Ready to initiate the calibration. Open the cell imaging software on the computer. Ensure that the microplate imager is turned on and the ports are connected to the computer.

Check the status bar in the bottom left of the screen to ensure that the temperature is set to 37 degrees Celsius and that the connection status should be highlighted in green as ready. Scan the plate barcode to initiate the imaging process. Provide a name to the cell plate and hit Save.

Brightfield and fluorescent images will be saved here. Click on Perform Brightfield Scan and, in the next screen, Plate and Scan Menu show the options for imaging. Before the assay, select Start Brightfield Scan.

Place the cell culture microplate and the plate cover on the tray holder and align well A1 with the A1 mark. Click on Close Tray. Brightfield image acquisition appears in the next screen with a plate map.

Click on Scan All Wells and scan for 30 to 35 minutes. On completion of the calibration, the software displays the Load Cell Plate dialogue box. Then, click on Open Tray to replace the utility tray with a cell culture microplate.

Note that the cell culture plate is in the microplate imager. After the scan, remove the cell culture microplate and place it in the analyzer to perform the assay. Then, click on Load Cell Plate to initiate the assay.

Wait until the assay starts and display the estimated completion time. Upon completion of the assay, the software displays Unload Sensor Cartridge and Cell Plate dialogue box. Click on Eject and remove the cell culture microplate from the analyzer.

Carefully remove the sensor cartridge and replace the cell plate lid. The Assay Complete dialogue box appears. Then, click on View Results to open the assay result file or to normalize the data.

After the assay completion, scan the plate barcode with the handheld barcode reader. If the plate has already been imaged, it will not require a new name. Select Fluorescence and Cell Count.

Place the cell plate on the tray holder and click on Close Tray. In the image acquisition window, select Scan All Wells to begin imaging. Review the fluorescent images and cell counts in the imaging and cell imaging application by randomly clicking on a couple of the wells.

Once the fluorescence imaging is complete, export the images for additional references. Once the imaging and cell count is complete, open the results file and click on Normalize. Click on Import and select Apply for the desktop software to normalize the assay with cell count automatically.

The oligomycin A and FCCP stressor mix injection increased the control group's baseline activity. Stressors showed a significantly high energy level in the control and treatment one groups. On the other hand, treatment two had a comparatively lower baseline activity, and the cells became more aerobic.

The figure indicates both control and experimental cells uses glycolysis and mitochondrial oxidative phosphorylation for ATP generation. The experimental group displays a reduction in ATP generation via glycolysis and an increase in mitochondrial respiration. The basal respiration rate in the treatment group is comparatively less than the control group.

The respiration and ATP production rates in both groups are decreased, along with the proton leak followed by an oligomycin A injection. The respiration rates of the cells rise back again to their maximal respiration after the injection of FCCP. The final injection of rotenone and antimycin A decreases the OCR again.

After the third injection, the mitochondrial respiration is shut down by the combination of rotenone and antimycin A.Compared to the control group, the basal and maximal respiration of the treatment groups has relatively no change. The figure shows that the control group is highly dependent on the glucose pathway, while glutamine oxidation was efficient in the control group. The fatty acid pathway shows that the treatment has increased the overall capacity of the cells.

The availability of multiple injection ports on the acid plate helps us to determine the effect of multiple drugs on cellular energetics at a single time point.