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Cell death/survival signal by ceramide and sphingosin-1-phosphate

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Introduction Protocol References Credit lines
Category
Biosynthesis & Metabolism
Protocol Name

Cell death/survival signal by ceramide and sphingosin-1-phosphate

Authors
 Kazuyuki Kitatani
Division of Clinical Laboratory Medicine, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Tottori University

Makoto Taniguchi
Division of Clinical Laboratory Medicine, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Tottori University

Toshiro Okazaki *
Division of Clinical Laboratory Medicine, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Tottori University
*To whom correspondence should be addressed.
KeyWords
Reagents
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N-acetyl-D-sphingosine (C2-Cer) (Matreya #1901, Pleasant Gap, PA, USA)

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N-Hexanoyl-D-erythro-sphingosine (C6-Cer) (Matreya #1900)

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D-erythro-S1P (Matreya #1803)

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Ethanol (Wako Pure Chemical, Osaka, Japan)

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HL-60 cells

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RPMI 1640 medium (sigma) containing 10% fetal bovine serum (FBS)

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4', 6-Diamidino-2-phenylindole (DAPI) (Roche, Basel, Swizerland)

Instruments
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Fluorescent microscopy Leica DMRB (Leica microsystems, Wetzlar, Germany)
Methods
1.

Making stock solution of C2-Cer or C6-Cer, and their preservation

1) 

 Dissolve C2-Cer (10 mg) and C6-Cer (10 mg) in 1.46 ml and 1.25 ml of 100% ethanol and mixed, respectively.

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2) 

 Dispense as each 200 μl of these solutions to dark glass vial tube and dry under N2 gas.

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3) 

 Store vial tubes at -80°C.

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4) 

 Reconstitute Cer with 200 μl ethanol (final concentration: 20 mM).

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2.

Making stock solution of S1P and their preservation

1) 

 Dissolve S1P (5 mg) in 1.32 ml of phosphate-buffered saline (PBS) containing 0.4% fatty acid free BSA (PBS/BSA) (final concentration: 10 mM).

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2) 

 Sonicate the solution.

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3) 

 Dispense and store tubes at -80°C.

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3.

Detection of cell death by DAPI staining after treatment with C2-Cer and S1P

1) 

 HL-60 cells were seeded at a density of 3 x 105 cells/mL in 35 mm non-treated dishes in RPMI 1640 containing 2% FCS.

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2) 

 After 30 min, S1P (Final concentration: 0-20 mM), C2-Cer (Final concentration: 10 μM), or combination of both was added with various concentrations.

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3) 

 After 24 hr, cells were harvested, washed with PBS, and re-suspended in PBS.

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4) 

 The cells were attached on the slide glass by cytospin at 200 x g for 5 min.

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5) 

 The cells were fixed with PBS containing 1% paraformaldehyde for 15 min at 4°C.

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6) 

 After washing twice with PBS, cells were treated with DAPI (Final concentration: 2 μg/ml) in PBS for 20 min at room temperature.

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7) 

 Cells were washed three times with PBS, rinsed with distilled water, and amounted with cover glass.

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8) 

 Condensation of DNA was detected by fluorescent microscopy using a UV-filter (excitation at 365 nm, emission at 480 nm).

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9) 

 At least 200 cells for determination were counted under a fluorescent microscope, and the cells with nuclear condensation and fragmentation were expressed as dead cells.

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Date of registration：2011-09-05 16:19:40
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