SphingomyelinQuantitationPostcholineLabelingofHL60Cells

Lipid Extraction

1) Following the appropriate time of treatment, transfer 4.5 ml into each of two duplicate glass pyrex tubes and maintain on ice.

2) Spin down cells in the table top centrifuge at 1,200 rpm, 40C, for 5 minutes.

3) Gently aspirate and discard the spent media (remember, consider it radioactive). Be extremely careful not to disturb the pellet since it is somewhat dispersed.

4) Immediately upon aspirating each tube, add 3 mls of chloroform-methanol, 1:2, and vortex vigorously!!

--> It is necessary to add the C:M immediately in order to get good dispersion of the pellet. If too much time passes prior to resuspension, the pellet does not dissolve well and sonication is necessary.

--> This is a good point to stop by storing the samples in the -200C freezer.

5) Add 0.8 ml of water and vortex vigorously.

--> Still have a monophase and it may be best to allow this monophase to equilibrate for a little while.

6) Spin out all large debris by spinning at 3,000 rpm for 5 mins.

7) Transfer the supernatant to a new pyrex glass tube and vortex again. Do not transfer any of the pellet, even if it is necessary to sacrifice some of the sample.

8) Add 1 ml of chloroform to break phases and vortex vigorously.

9) Add 1 ml of water and again vortex vigorously.

10) Spin samples at 3,000 rpm for 5 mins. to completely separate phases.

11) Aspirate off the top phase and the interface.

--> Remove enough such that no aqueous material remains at the meniscus.

12) The lower phase should constitute ~2 ml. Transfer 1.5-1.7 ml of this to a new pyrex glass tube.

--> This is a good point to stop by storing the transferred samples in the -200C freezer.

SMase assay

13) Dry down samples completely.

14) Resuspend dried lipids in 50 µl TMT buffer.

15) Vortex vigorously and sonicate via three 1 minute bursts, vortex and rest for 1 minute in between.

16) Pre-incubate tubes in 37°C water bath for 5 minutes.

17) Add 50 µl of SMase solution to each reaction tube and allow reaction to proceed for 30 minutes.

--> Remember to have a control tube containing no enzyme to determine background.

18) Add 1.5 ml Chloroform/Methanol (2:1) to each tube to stop reactions.

19) Vortex vigorously and add 0.2 ml water to complete the Folch extraction.

20) Vortex tubes and spin down for 5 minutes at 1,000Xg (break on low).

21) Collect ~400 µl of the upper phase into previously prepared plastic scintillation vials and count samples.