Ud 100 sonicator

To analyze the lipids accumulating in cells, samples were prepared as follows. Cells were collected from the main cultivation and freeze-dried. Twenty milligrams of dried cells were transferred into a 15-mL glass tube, to which 2 mL of a solvent (chloroform:methanol = 2:1) was added, and lipids extracted using the UD-100 sonicator (TOMY, Tokyo, Japan: set at an output of 70 for 10 min). The resultant samples were left overnight in a cool dark place to improve extraction. Subsequently, they were centrifuged at 3,000 × g for 10 min and the supernatants were collected. To wash the samples, equal amount of 0.9 % aqueous KCl solution was added to the supernatant, mixed, and subjected to centrifugation at 2,000 × g for 5 min. Approximately 2 mL of the bottom fraction was collected as the organic layer, transferred to a 15-mL glass tube, and placed in a rotary vacuum evaporator (V-850: BUCHI, Flawil, Switzerland). The resultants were dissolved in 200 μL chloroform, and eventually used for TLC. A 4-μL aliquot of each sample was spotted onto the origin (ori) of a silica gel plate (#60F254: Merck Millipore, Darmstadt, Germany). Lipids on the plate were developed with an eluent (hexane:diethylether:acetic acid = 80:20:1) and stained with 5 % (v/w in EtOH) phosphomolybdic acid solution (#32186-00: Kanto Kagaku Co., Ltd., Tokyo, Japan).