| S Preparation
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C57BL/6 mice were purchased from CLEA Japa … C57BL/6 mice were purchased from CLEA Japan and maintained according to the Guidelines for Animal Experimentation of Tohoku University.
Mouse tissues (approximately 100 mg) were placed in 1.5 ml siliconized sample tubes. Then, nine volumes of acidic methanol (pH 4.0), 1 μM (final concentration) of 17:0-LPA, and 10 μM (final concentration) of 17:0-LPC were added to the tube. The obtained mixture was homogenized for 10 min by Micro Smash MS-100R (TOMY) (3,000 rpm at 4°C). After an initial centrifugation at 1,000 g for 10 min at 4°C, the supernatants were further centrifuged at 21,500 g for 10 min at 4°C. The resulting supernatant was passed through a filter (0.2 μm pore size, 4 mm inner diameter; YMC), and 10 μl of the filtrate was subjected to LC-MS/MS.
To obtain a serum sample, a blood sample was collected with a noncoated capillary (910-01-75; Hirschmann Laborgerate), incubated at 37°C for 1 h, allowed to stand at 4°C for 12 h, and centrifuged at 1,500 g for 10 min at 25°C. Plasma was collected with a heparinized capillary (1-040-7500-HC; Drummond), followed by addition of 1 mM of EDTA (final concentration), and centrifuged at 1,500 g for 10 min at 25°C. The plasma and serum samples (10 μl each) were placed in 1.5 ml siliconized sample tubes, deproteinized by mixing with 90 μl acidic methanol (pH 4.0), homogenized for 3 min in an ultrasonic bath, and centrifuged at 21,500 g for 10 min at 4°C (ice-cold water). The supernatants were filtered and 10 μl of the filtrate was subjected to the LC-MS/MS. he filtrate was subjected to the LC-MS/MS.
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