Metabolic labeling of sphingolipids

 Pre-culture the B16 melanoma cells in minimum essential medium (MEM) supplemented with 10% FBS, 100 μg/ml of streptomycin, and 100 units/ml of penicillin in a CO₂ incubator.

 Culture the cells described in step 1-1) in a 6-well plate (1 x 10⁵ cells/well) overnight.

 Add 1 μCi of [¹⁴C]serine to each well.

 Incubate at 37°C for 24 h in a CO₂ incubator.

 Harvest the cells using a cell scraper.

 Centrifuge the cells at 700 x g for 5 min at 4°C, wash with ice-cold PBS,and then re-suspend in 750 μl of i-propanol, hexane, and water (55/35/10, v/v/v).

 Sonicate the suspension for 20 min in a sonic bath.

 Centrifuge at 10,000 x g for 3 min.

 Withdraw the supernatant and transfer it to a new tube.

 Dry the sample with a centrifugal concentrator.

 Dissolve the dried material in 20 μl of chloroform and methanol (2/1, v/v).

 Spot the sample on a Silica Gel 60 TLC plate and perfom two-dimensional TLC as described in step 1-13~15).

 Develop the TLC plate with chloroform, methanol, formic acid, and H₂O (65/25/8.9/1.1, v/v/v/v) (1^st run).

 Dry the TLC plate with a hair-dryer. Rotate it 90° and develop with chloroform, methanol, and 5 N NH₄OH (50/40/10, v/v/v) (2^nd run).

 Dry the TLC plate with a hair-dryer. Develop the plate with diethylether in the opposite direction to the second run to separate ceramides from other neutral lipids.

 Dry the TLC plate with a hair-dryer. Put the TLC plate onto an imaging plate of the exposure cassette.

 Expose the plate overnight and use an imaging analyzer.

 Culture the CHO cells in Ham’s F-12 medium supplemented with 10% FBS, 100 μg/ml of streptomycin, and 100 units/ml of penicillin in a CO₂ incubator.

 Culture the cells described in step 2-1) in a 6-well plate (1 x 10⁵ cells/well) overnight.

 Add 0.5 μCi of [³H]sphingosine to each well.

 Incubate at 37°C for 24 h in a CO₂ incubator.

 Harvest the cells using a cell scraper.

 Centrifuge at 700 x g for 5 min at 4°C, wash with ice-cold PBS, and then re-suspend in 100 μl of ice-cold water.

 Add 375 μl of chloroform, methanol, and HCl (100/200/1, v/v/v) to the cell suspension, and mix well with a Voltex mixer.

 Add 125 μl of chloroform and 125 μl of 1% KCl, and mix well with a Voltex mixer.

 Centrifuge at 10,000 x g for 5 min, withdraw the lower phase (organic phase), and transfer it to a new tube.

 Dry the sample with a centrifugal concentrator.

 Dissolve the sample in 20 μl of chloroform and methanol (2/1, v/v).

 Spot the sample on a Silica Gel 60 TLC plate and develop it with 1-butanol, acetic acid, and water (3/3/1, v/v/v).

 Dry the TLC plate with a hair-dryer and spray the TLC with an enhancer EN3HANCE.

 Expose the TLC plate to X-ray film for at least 2 days at -80°C. Visualize the radioactive spot with a film processing system.

 Culture the yeast cells overnight in YPD medium.

 Dilute the culture with SC medium lacking serine to 0.5 A ₆₀₀ units/ml and culture for an additional 5 h.

 Collect the cells by centrifugation at 1,500 x g for 3 min, and re-suspend in 500 μl of fresh SC medium lacking serine to 2.0 A₆₀₀ units/ml.

 Add 0.5 μCi of [¹⁴C]serine to the cell culture.

 Incubate for 1 h at 30°C with gentle shaking.

 Harvest the cells by centrifugation at 1,500 x g for 3 min at 4°C and then wash with 1 ml of ice-cold water.

 Add 150 μl of ethanol, water, diethyl ether, pyridine, and 15 N ammonia (15/15/5/1/0.018, v/v/v/v/v) to the cell pellets.

 Mix the sample with a Voltex mixer.

 Incubate at 60°C for 15 min.

 Centrifuge at 10,000 x g for 1 min and withdraw the supernatant.

 Add the solvent to the residue and extract the sphingolipids again as described in step 3-7).

 Mix the supernatant obtained in step 3-10) and 11).

 Dry the supernatant with a centrifugal concentrator.

 Dissolve the dried sample in 20 μl of chloroform, methanol, and water (5/4/1, v/v/v).

 Spot the sample on a Silica Gel 60 TLC plate and then develop with chloroform, methanol, and 4.2 N ammonia (9/7/2, v/v/v).

 Dry the TLC plate, expose it to an imaging plate, and then use an imaging analyzer.