This technique uses instruments commonly found in most laboratory environments and allows for the analysis of several samples simultaneously. This method can provide key insights into how lipid metabolism enzymes are regulated in a cellular context and in different genetic backgrounds. Inoculate a colony from a yeast culture plate into 20 milliliters of SC media containing 2%dextrose and incubate at 30 degrees Celsius for overnight with shaking at 200 rpm.
On the next day, dilute the culture in 50 milliliters of fresh media to a final OD of 0.2 and grow it for 24 hours until the stationary phase is reached. Prepare two 20 milliliter aliquots of quenching buffer for each sample and store them at minus 80 degrees Celsius. Simultaneously, prepare radiolabeling media by adding acetic acid sodium salt to dextrose free SC media at a final concentration of 10 microcurie per milliliter.
Collect the cells by centrifuging at 4, 100 x g for 10 minutes. Remove the supernatant and wash the cell pellet once with 20 milliliters of dextrose free SC media. Collect the cells again by centrifuging at 4, 100 x g for five minutes and transfer them to a labeled two milliliter microcentrifuge tube using dextrous free media.
Centrifuge the cells for another two minutes. Resuspend the cells in 500 microliters of dextrose free SC media and begin the radiolabeling period by quickly adding 500 microliters of radiolabeling media to each cell suspension. Incubate the tubes in a rotating incubator at 30 degrees Celsius for 20 minutes.
Meanwhile, pre cool the centrifuge equipped for 50 milliliter conical tubes to minus 10 degrees Celsius and thaw one 20 milliliter aliquots of quenching buffer. Once the radiolabeling period has ended use a pipette to plunge the entire one milliliter sample into thawed quenching buffer on ice. Collect the cell pellet by spinning in a pre cooled centrifuge at 5, 000 x g for three minutes and add 20 milliliters of fresh cold quenching buffer.
Vortex and shake the samples to fully resuspend them in quenching buffer and centrifuge the samples again to collect the cells. Once the cells are pelleted, thoroughly remove all quenching buffer from the samples by pouring off the supernatant and removing the excess with a pipette. Store the tubes at minus 80 degrees Celsius for further processing.
Weigh 0.3 grams of acid washed glass beads for each sample and store them in two milliliter microcentrifuge tubes on ice. Remove the cell pellets from the minus 80 degree freezer and place them on ice. Then, add 350 microliters of methanol and 700 microliters of chloroform to each sample and resuspend the cells.
Transfer them to micro centrifuge tubes containing pre-weighed glass beads. Lyse the cells by vortexing three times for one minute, with 30 second incubation on ice between agitations. Pour the cells into a 15 milliliter glass centrifuge tube and label the tube as tube A.Wash the microcentrifuge tube with one milliliter of methanol, vortex it, and pour the methanol into tube A.Add two milliliters of chloroform followed by 400 microliters of water to tube A for a final sample volume of 4.45 milliliters.
Vortex the samples for one minute followed by a five minutes centrifugation at 1000 x g to clearly separate the aqueous and organic phases with cell debris lying at the interface. Using a glass pasture pipette, collect the organic phase into a new glass centrifuge tube labeled B and add one milliliter of one molar potassium chloride. Add one milliliter of methanol and two milliliters of chloroform to tube A and repeat the vortexing and centrifugation steps.
Vortex and centrifuge tube B to separate the aqueous and organic phase. Remove the upper aqueous layer and collect the entire bottom organic layer into a four milliliter glass vial. Completely evaporate the solvent from the lipid extracts by drying under inert gas and preheat an oven to 145 degrees Celsius for heating the TLC plate.
Determine relative amounts of radiolabel taken up by the cells before loading them onto the TLC plate. Reconstitute the sample lipids in 40 to 50 microliters of chloroform and methanol mixed at a one-to-one ratio by vortexing for five minutes. Prepare 101 milliliters of the mobile phase solvent in a glass graduated cylinder.
Then, pour the solvent into a glass TLC chamber containing a 20 by 20 TLC saturation pad and a tight fitting lid. Prepare a channeled 20 by 20 silica gel 60G plate and mark a line 1.5 centimeters above the bottom of the plate using a pencil. Once the plate has been sufficiently heated and the TLC saturation pad is saturated with solvent, remove the TLC plate from the oven and immediately proceed to loading it.
Using a pipette, spot five microliters of sample onto the origin of each lane located 1.5 centimeters above the bottom of the TLC plate and repeat loading until 20 to 40 microliters of sample has been loaded into each lane. Once the standard and the samples have been loaded, place the plate in the developing chamber and wait until the solvent has reached the top of the plate. When the plate is fully developed, remove it from the chamber and allow it to dry in the fume hood for 20 minutes.
After the plate is dried, cover it with plastic film and place it in a developing cassette with an autoradiography screen. Remove the screen from the developing cassette and place it inside of a phosphor imager. Spray the TLC plate with p-Anisaldehyde reagent until the silica is saturated, then bake it in a 145 degree oven for five minutes or until bands have appeared.
To quantify lipid species, scrape the silica gel containing individual lipid species and transfer them to glass scintillation vials. Add six milliliters of scintillation fluid and vortex vigorously until the silica band has been reduced to small pieces. Place the rack with the scintillation vials into a scintillation counter and count by adjusting the counting time to two minutes per vial.
Phosphor imaging allows for visualization of autoradiogram of label-free fatty acid, triacylglycerol, diacylglycerol, cholesterol, and squalane. It was demonstrated that purified lipid species can be separated in this method and subsequently visualized by spraying of the TLC plate with p-Anisaldehyde reagent. Visualized species include all previously mentioned lipids in addition to sterile esters.
By applying a 10 minute chase period in radiolabel-free media following the pulse, it was observed at the major pool of squalane disappeared and total cholesterol was elevated. Similarly, the appearance of diacylglycerol in the chase period correlated with a decrease in the free fatty acids signal. Prior to performing the full protocol, it is important to determine whether the cells will uptake the acetic acid radiolabel in the growth condition and genetic background of interest.
