The overall goal of the following experiment is to test the functional status of mitochondria in living cells and isolated mitochondria by measuring multiple parameters of oxidative metabolism using the XF 96 extracellular flux analyzer. These parameters are basal respiration, A TP turnover, proton leak, and maximal respiration or spare respiratory capacity. This is accomplished by sequentially adding specific modulators of mitochondrial respiration to C two C 12 myoblast cells, and measuring the change in oxygen consumption rate, or OAR, an indicator of respiration, the conversion of glucose to lactate, which generates a TP through glycolysis.
The other major energy pathway in cells is also measured as ECAR or extracellular acidification rate. Mitochondrial profiling results can be obtained that provide insight into the central role that mitochondrial function plays in determining the lifespan of cells. Hello, my name is David Nichols.
I've been a mitochondrial artist all my career and the most important piece of equipment I've used in my studies has been the oxygen electrode. If you can measure the rate of oxygen uptake under different conditions with isolated mitochondria, you obtain a massive important information. When you move to cells, especially cells which are cultured in a multi-well plate or on a cover slip, classical methods don't work.
This is a major problem, which was overcome a few years ago by the development of a multi-well plate assay enabling us to study the respiration of cells in parallel in a multi-well plate format. What you're going to see today is a very simple but information rich experiment, which we've devised in order to monitor basic mitochondrial bioenergetic function in cultured cells To Begin seed cells. In XF 96 cell culture plates with 10, 000 cells per well in 100 microliters of growth, medium and place in a 37 degree Celsius incubator with 10%carbon dioxide.
The cells can be assayed in the XF 96, 24 hours after seeding using the assay. Wizard generat a template with the following group layout. Columns one through 12 all contain 10, 000 C two C 12 cells.
Group one in blue are used as control cells. Group two in red will be administered the drugs oligo Mycin, FCCP, and roone. Next, prepare the compounds in X-F-D-M-E-M assay medium as described in the written portion of the protocol.
Oligo Mycin, FCCP, rotten known. These compounds are pre dispensed in the XF cell MIT stress test kit. Using the XF prep station, replace the growth medium with DMEM running medium.
Finally incubate the cells in a 37 degree Celsius incubator without carbon dioxide for 60 minutes to allow cells to pre equilibrate with the assay Medium prior To adding the injectable compounds into the sensor cartridge form the compounds to 37 degrees Celsius. Then load them into the injector ports as follows. For columns one through four, load 16, 18, and 20 microliters of XF assay medium or DMEM into ports A, B, and C respectively.
For columns five through 12, load 16 microliters of all liga mycin into port A 18 microliters of FCCP into port B, and 20 microliters of rot known into Port C.Next, define the protocol Commands set loop to three times. Mix to three minutes and measure to three minutes. Next, click on start.
Follow the instructions on the screen that will instruct the user when to load the cartridge when the calibration is complete. Follow user prompts and add the cell plate for the experiment. Then click continue and allow the assay to run.
Remove the plate when the experiment has ended. This Assay is derived from the classic experiment to profile mitochondrial function and serves as a framework with which to build more complex experiments aimed at understanding various compound effects on cell metabolism, mitochondrial function, and overall bioenergetic activities. Here we observed the expected response in oxygen consumption rate as the cells were treated with each success of compound.
For liga Mycin Oard decreased as a result of blocking a TP synthesis at mitochondrial complex five. Since the cells were unable to synthesize a TP via oxidative phosphorylation after a liga mycin treatment, they reverted to glycolysis to meet their demand for a TP.Thus, we observed an increase in ECAR, the extracellular acidification rate, an indicator of glycolysis as shown previously. FCCP acts as an uncoupling agent and removes the limitation of proton reentry.
Respiration accelerates and is now limited by electron transport. Finally, rote known inhibits mitochondrial complex one, which causes the flow of electrons to cease in the electron transport chain, and thus the consumption of oxygen was drastically reduced. Here information is obtained about the basal respiration of the cells, the percent of oxygen consumption devoted to a TP production, as well as the amount devoted to maintaining the proton gradient due to proton leak.
Furthermore, we may obtain the maximal respiratory rate under conditions of uncoupled respiration, sometimes referred to as spare respiratory capacity. And finally, we can determine the amount of oxygen consumption not due To mitochondrial processes. You've just seen with the seahorse.
XF 96, a series of experiments that give a tremendous amount of information on the BIE energetics of the mitochondria in the cell. The basic experiment, which is so important, we call it experiment number one, gives you information on basal respiration, a TP turnover, mitochondrial proton leak, and spare respiratory capacity. These parameters give you this fundamental information on the bioenergetic status of the mitochondria in the cell.
