Research into lipid droplet biology has been hampered by difficulty of purification. Additionally, studying cellular lipid flux requires simultaneous purification of multiple organelles, and no simple protocols were currently available. The main advantage of this technique is to make lipid droplet purification more accessible.
We modified a commercially available kit to simultaneously isolate lipid droplets, endoplasmic reticulum, and lysosomes from a single sample. Liver lipid droplet accumulation predisposes obese and alcoholic patients to progressive liver disease. This has shifted the view of lipid droplets from inert storage compartments to dynamic biologically important organelles.
To begin, use sterile forceps and dissection scissors to cut the skin and muscle layers of the abdomen of the euthanized mouse on the posterior-anterior axis, exposing the liver. Cut the ligaments connecting the liver to the intestine. Using forceps, pull the intestine away from the liver, toward the posterior.
Cut the ligament connecting the liver to the diaphragm. Then, cut between the liver and the dorsal ribcage, moving from anterior to posterior, until the liver is liberated. Transfer the liver to a cold five-centimeter Petri dish with 10 milliliters of cold 1x PBS, and wash the liver twice on a rocking platform in 10 milliliters of cold 1x PBS for five minutes at four degrees Celsius.
After the final wash, pour off the 1x PBS, and use a size-10 sterile surgical blade to dice the liver into three-to five-millimeter pieces within the dish. Then, wash the diced liver two times with 10 milliliters of cold 1x PBS for five minutes at four degrees Celsius. Decant the 1x PBS, and transfer the diced liver pieces to a cold 45-milliliter Dounce homogenizer, ensuring all pieces are at the bottom.
Add seven milliliters of cold 1x IE buffer. Homogenize on ice by moving the pestle up and down 20 times, making sure that the pestle goes to the bottom of the homogenizer during each stroke. Transfer the homogenate to a cold 15-milliliter centrifuge tube.
Rinse the homogenizer with one milliliter of cold 1x IE buffer, and add it to the rest of the homogenate. To remove nuclei, centrifuge the homogenate in a high-capacity centrifuge at 1, 000 times g for 10 minutes at four degrees Celsius. Ensure that any lipid gathered at the top of the 15-milliliter tube is resuspended to prevent LD loss.
Making sure that nuclear pellet at the bottom of the tube is undisturbed, transfer the lipid-containing post-nuclear supernatant to a fresh cold 50-milliliter centrifuge tube. Transfer a 100-microliter aliquot of the post-nuclear supernatant to a cold 1.7-milliliter microcentrifuge tube on ice for later purity analysis. To remove mitochondria, centrifuge the post-nuclear supernatant sample in a refrigerated high-speed centrifuge at 12, 000 times g for 15 minutes at four degrees Celsius.
Ensure that any lipid gathered at the top of the tube is resuspended to prevent LD loss, and then transfer the post-mitochondrial supernatant to a 13-milliliter ultracentrifuge tube. Transfer a 100-microliter aliquot of the post-mitochondrial supernatant to a cold 1.7-milliliter microcentrifuge tube on ice for later purity analysis. Fill the ultracentrifuge tube with post-mitochondrial supernatant to the brim with cold 1x IE buffer, to prevent it collapsing during ultracentrifugation.
Balance the samples, and then centrifuge in an ultracentrifuge at 100, 000 times g for 60 minutes at four degrees Celsius. To remove the LD layer, tilt the tube at a 45-degree angle, and aspirate with a glass pipette. After transferring the pellet to a cold 1.7-milliliter microcentrifuge tube, collect 100 microliters of the post-ER supernatant under the lipid layer for purity analysis.
To pellet any cell debris, centrifuge in a microcentrifuge at top speed for five minutes at four degrees Celsius. Then, warm the tube gently with hands to help resuspend the LD layer, and transfer the supernatant, including the lipid layer, to a new 1.7-milliliter microcentrifuge tube. Repeat these washing steps, warming the tube two to three times, until the pellet is no longer visible.
Then, to wash the pellet-free LD supernatant, add 1x PBS to a final volume of 1.5 milliliters, and centrifuge in a microcentrifuge at top speed for five minutes at four degrees Celsius. Use a glass pipette to remove one milliliter of 1x PBS, which is underneath the lipid layer, without disturbing the lipid layer. Repeat LD washing four to five times, until the 1x PBS is visually transparent with no turbidity.
Then, use a glass pipette to remove all 1x PBS below the lipid layer, and use the resulting pure LD fraction for downstream analysis. When representative 20x fluorescent and brightfield LD images were taken from both the ER kit LD isolation and the sucrose LD isolation methods, they revealed similar levels of particulate matter, suggesting similar purities using two different protocols. Following purification, LD triglyceride and protein levels were measured using colorimetric assays, and the data were normalized to liver weight, showing that when the starting material was normalized, LD triglyceride and protein yields were similar using the ER kit and the sucrose method.
LD diameters were quantified using image analysis software, showing that the ER kit LD isolation and sucrose LD isolation yielded LDs of similar sizes. To assess LD purity, samples were assayed by immunoblotting, showing that the LDs derived from the ER kit LD isolation and the sucrose gradient protocol both had equal purity. PLIN2, an LD marker, was detected in all samples except for the ER kit LD isolation PER and the sucrose's PNS.
Purity analysis of an ER fraction using immunoblots shows that they were free of LD marker PLIN2 but had significant levels of the ER SEC61A protein. Purity of a lysosomal fraction after further purification of lysosomes using the lysosome enrichment kit was also assessed. Discovery is currently the most useful application of our protocol.
We perform lipidomics in purified organelles, and showed lipotoxins are increased specifically in lipid droplets in alcoholic liver disease.
