Extraction of sphingosines ceramides and sphingomyelins

Extraction of sphingosines, ceramides, and sphingomyelins is most easily accomplished via two commonly used methods. The most prevalent is the method of Bligh and Dyer (6). Also of use is the Folch extraction (7). The two methods rely upon the hydrophobic/organic nature of the sphingolipids to separate them from other cellular components (Figure 3). The resulting

Water Chloroform

Aqueous

Organic w w

Figure 3. Illustration of the phase break induced in the Bligh and Dyer extraction by the addition of water and chloroform. To obtain clear phases allow to sit or spin in a table-top centrifuge at 2000 g. The organic phase may be collected by first aspirating the aqueous phase, then collecting it, or by directly taking the organic phase.

extract can be used for analysis of sphingolipids using various protocols and these measurements can be normalized to lipid phosphate.

Protocol 1. Extraction of sphingolipids by the method of Bligh and Dyer (6)

Reagents

• chloroform/methanol (1:2 pre-mixed) • dry nitrogen

Method

1. Grow 2-5 x 106 cells per sample to be analysed. Treat as desired. Adherent cells need to be suspended.

2. Pellet cells by table-top centrifugation of 500 gfor 5 min.

3. Aspirate off the media or wash PBS from the pellet.

4. Resuspend the cell pellet in 3 ml of chloroform/methanol (1:2 pre-mixed) and vortex until an even suspension is gained.

5. Add 0.8 ml of water and vortex. Transfer the resuspended cells to a glass screw-cap tube and set on the bench for 30 min or overnight at 4°C for best extraction. If there is a phase break at this point it can be corrected by the addition of 0.5 ml of methanol. A premature phase break can hinder proper extraction.

6. Pellet cellular debris via table-top centrifugation at 2000 g for 5 min. Transfer liquid material to a fresh tube and discard cellular debris.

7. Add 1 ml of chloroform and 1 ml of water and vortex. These additions will induce a break of the liquid material into an organic (lower) and aqueous (upper) phases (Figure 3). Allow 15-30 min for the phases to separate and then centrifuge for 5 min at 2000 g to obtain clean phase separation.

Protocol 1. Continued

8. One may now collect the lower organic phase directly or aspirate the aqueous phase and then collect the organic phase.

9. The extracted lipids should be in the 2 ml of chloroform and should be dried down via a speed vacuum apparatus or under a stream of dry nitrogen.

10. The dried down lipids should now be resuspended in chloroform with one aliquot designated for phosphate measurement (usually one-third) and another aliquot for the experimental measurement desired (usually one-third; the last third is a back-up).

Protocol 2. Extraction of sphingolipids by the method of Folch

Reagents

Method

1. Grow 2-5 x 106 cells per sample to be analysed. Treat as desired. Adherent cells need to be suspended.

2. Pellet cells by table-top centrifugation of 500 gfor 5 min.

3. Aspirate off the media or wash liquid from the pellet.

4. Add 1 ml of methanol to the pellet, vortex, and transfer to a glass screw-cap tube.

5. Add 2 ml of chloroform to the above and vortex. Make sure that the cell pellet is completely resuspended. For best extraction, let samples sit on the bench for 30 min or at 4°C overnight.

6. Pellet cellular debris via table-top centrifugation at 2000 g for 5 min. Transfer liquid material to a fresh tube and discard cellular debris.

7. Add 0.8 ml of water and vortex. This addition should induce a phase break in the samples to an organic (lower) and aqueous (upper) phases.

8. One may now collect the lower organic phase directly or aspirate the aqueous phase and then collect the organic phase.

9. The extracted lipids should be in the 2 ml of chloroform and should be dried down via a speed vacuum apparatus or under a stream of dry nitrogen.

10. The dried down lipids should now be resuspended in chloroform with one aliquot designated for phosphate measurement (usually one-third) and another aliquot for the experimental measurement desired (usually one-third; the last third is a back-up).

Once the extraction and aliquoting of lipids is accomplished the actual analysis of each sphingolipid class can begin. Storage of the lipids should be as either a powder or in chloroform at -20 °C. As noted in step 10 of either protocol, you should end up with three tubes, each containing one-third of your extract. Both methods are useful for the extraction of the three classes of sphingolipids, but are not efficient for sphingoid phosphates.

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