To begin, seed the 143B human osteosarcoma cells in a six-well plate. Once cells become 75%confluent, replace the medium in each well with the medium containing 10 millimolar 13C4 aspartate, and incubate for eight hours to achieve a steady state for labeling the cells. For isolating metabolites, cool the prepared buffer overnight at minus 80 degrees Celsius or place it on dry ice.
Meanwhile, take a plate from the incubator and aspirate the medium from each well using a pipette. Wash it with PBS twice, and then remove the residual PBS before proceeding further. Now place the plate on dry ice and add 800 microliters of prepared buffer to each well.
To facilitate cell lysis, incubate the plate for 15 minutes at minus 80 degrees Celsius. After incubation, scrape each well on dry ice using a cell lifter, and transfer the lysate to a 15 milliliter microcentrifuge tube placed on dry ice. Vortex the lysate for 10 minutes at four degrees Celsius, and centrifuge it at four degrees Celsius and 17, 000 g for 10 minutes.
Then transfer the supernatant to a 1.5 milliliter microcentrifuge tube and dry it in a four degree Celsius vacuum concentrator with a cold trap under a high vacuum for approximately six hours. Store the dried metabolite pellet at minus 80 degrees Celsius until further use. For analyzing the sample using liquid chromatography mass spectrometry or LCMS, add 100 microliters of HPLC grade water to the dried pellet and vortex as demonstrated.
Centrifuge the samples at four degrees Celsius for 10 minutes at maximum speed. Then transfer 25 microliters of supernatant to the LCMS vial, and inject two microliters into the LCMS system. To perform liquid chromatography, use a constant flow rate of 0.15 milliliter per minute, under the gradient mode of a mobile phase B from 80 to 20%for 20 minutes, 20 to 80%for 0.5 minutes, and hold at 80%for 7.5 minutes against mobile phase A.Next, perform mass spectroscopy by choosing a full scan between mz 70 to 1, 000 dalton.
And resolution at 70, 000. Set AGC target of one times 10 to the sixth, and a maximum injection time of 20 milliseconds. Next, set the runtime to 16.5 minutes.
Polarity set to positive. AGC target set to one to the power of five. Maximum IT set to 20 milliseconds.
And scan range set to 70 to 1, 000 mass-to-charge ratio. Then set the spray voltage at 3.0 kilovolts. And the heated capillary at 275 degrees Celsius.
Select the sheath gas flow at 40 units, the auxiliary gas flow at 15 units, and the sweep gas flow at one unit. For 13C5 glutamine isotope tracing, once the cells reach 75%confluency, change the medium to two millimolar 13C5 glutamine-containing medium, and incubate to achieve a steady state of labeling the cells of interest. Isolate and analyze the metabolites as previously demonstrated.
After isolating and analyzing the metabolites, analyze the succinate dehydrogenase or SDH activity by calculating the percent labeling of 13C3 fumarate, 13C4 fumarate, 13C3 succinate, and 13C4 succinate. Then evaluate succinate oxidation and fumarate reduction using the formula. In vehicle-treated conditions, the SDH complex favored the forward activity, and the incorporation of 13C4 succinate into 13C4 fumarate was higher than 13C3 fumarate into 13C3 succinate.
In antimycin-treated osteosarcoma cells, the SDH complex favored the reverse activity, and the incorporation of 13C3 fumarate into 13C3 succinate was more significant than 13C4 succinate into 13C4 fumarate.
