It's a messer that call answer a key question that you have in also a function of field such as ATP level and the oxygen consumption rates. The new or the one page old, this technique is that it's easy to perform and it's a lot for a special evaluation of cellular mitochondrial function. To start this experiment, seed 20, 000 HepG2 cells in a glass bottom Petri dish.
Add beta-hexachlorcyclohexane or B-HCH in incubator at 37 degrees Celsius and five percent CO2 for 24 hours. Prepare one millimolar mitochondrial green fluorescence probe solution by adding anhydrous DMSO to DMEM cell culture medium. Then add one milliliter per dish of this solution to the cells in glass bottom Petri dishes.
And incubate at 37 degrees Celsius for 45 minutes. After incubation, remove the mitochondrial green fluorescent probe solution from the cells. Add one milliliter per dish of freshly prepared at 37 degrees Celsius DMEM cell culture medium.
To detect green fluorescence in mitochondria, observe the cells under a confocal microscope. To evaluate levels of cellular ATP, seed to 20, 000 HepG2 cells in six well plates. Add beta-HCH and incubate at 37 degrees Celsius for 24 hours.
Add 200 microliters of lysis buffer to the cells in each well. Centrifuge at 12, 000 times G for five minutes at four degrees Celsius, and proceed with collecting supernatant as described in the text protocol. Add 100 microliters ATP detection buffer to the plate.
Add 100 microliters of collected cell supernatant to a 96 well plate at room temperature and proceeded to detect cellular ATP by a luminometer as described in the text protocol. For assessment of mitochondrial membrane potential, grow HepG2 cells in six well plates with the addition of beta-HCH, as previously described. Collect the cells by digesting them with 0.5 milliliters 0.25%EDTA for one minute at 37 degrees Celsius.
Add one milliliter DMEM to stop digestion and transfer the digested cells to 1.5 milliliter tubes. Centrifuge the tubes at 1000 times G for three minutes, and then discard the supernatant. Dilute the cell pellet with 0.5 milliliters of cell culture medium and 0.5 milliliters JC1 mitochondrial membrane potential dye.
And incubate for 20 minutes at 37 degrees Celsius. Next, centrifuge the tubes at 600 times G for three minutes at four degrees Celsius, and then wash the cell pellet according to the text protocol. After centrifuging the washed pellet, remove the supernatant.
And re-suspend the pellet in 0.5 milliliters of JC1 dyeing buffer. Analyze the JC1 stained cells via flow cytometry to detect green and red fluorescence. When the JC1 monomer is detected, set the excitation light to 490 nanometers and the emission light to 530 nanometers.
When the JC1 polymer is detected, set the excitation light to 525 nanometers and the emission light to 590 nanometers. To analyze the oxygen consumption rate, seed HepG2 cells in a 96 well cell culture plate in a density of 4, 500 cells per 100 microliters per well. After 24 hours of incubation at 37 degrees Celsius, make sure that the cells in each well are covered with medium.
Before running the OCR software, load the microplate with cartridge and the cell culture plate into an extracellular flux analyzer as described in the text protocol. Open the OCR software and in the apps drop down menu, choose cell mito stress test kit, and click on the start app button. Next, click on the run stress test button.
When the cell stress test screen appears, enter the number of cells seeding per well in the cell seeding number box and the average OCR in the average at basal cell OCR box. Enter the final working concentration for each reagent and then inject the reagents. Click on the next button.
When the group info screen appears, assign a group to unassigned wells by choosing a color and giving a name. Then click on the appropriate wells. Click the next button and in the stress test injection layout screen that appears, click on start to run the stress test on the analyzer.
After the completed run, follow the prompts in the software and then remove and discard the cartridge and cell plate. Average intensity of mitochondrial green fluorescent staining decreased in HepG2 cells exposed to beta-HCH. This shows that there is a decrease in mitochondrial number and increase in damaged mitochondria consistent with the increase in pesticide concentration.
Furthermore, ATP levels in HepG2 cells decreased with the increased concentrations of beta-HCH exposure, showing the decreased function of mitochondria in these cells. After JC1 staining for mitochondrial membrane potential, or MMP, flow cytometer showed high red green JC1 fluorescence in intact HepG2 cells. This was due to the presence of red fluorescent JC1 aggregates, which are a sign of high MMP.
In cells treated with beta-HCH, red green JC1 fluorescence decreased due to a presence of green fluorescent JC1 monomers, a sign of low MMP. Finally, oxygen consumption rate also decreased with the increased concentrations of beta-HCH exposure, further showing that this pesticide impairs proper mitochondrial function. Oh, well, attempt to this procedure, it's important though, to remember to see that cells as appropriate to test it.
After its development, this definitely paves a way for researches in the field of fundamental function to explore relevant organisms in the medical role associated with it. After watching this video, you should have a good understanding of how to detect an amount or quality, capacity, now member or potential under spoutage sheet function.
