- Cultured cells were scraped with 2 mL of ice-cold methanol, 100 pmol of PIP1(16:0/16:0), PIP2(16:0/16:0), and PIP3(16:0/16:0) were added as internal standards and 2 μL of 1 nmol/μL PIP2(8:0/8:0) was added as an adsorption protectant.
- 2mL of 1 N HCl and 1mL of water were added to suspension. The solution was shaken well.
- 0.15 mL of 2 M NaCl and 2 mL of chloroform were added to suspension, followed by vigorous shaking for 3 min while cooling with ice.
- Samples were centrifuged at 1000g for 5 min, and the lower layer was collected.
- After addition of 1.5 mL of methanol, each sample was applied to a DEAE-cellulose (Wako Pure Chemicals) column.
- Column-bound lipids were washed with chloroform/methanol (1:1) (3 × 1 mL), and chloroform/methanol/28% aqueous ammonia/acetic acid (200:100:3:1) (3 × 1 mL) in series.
- Highly acidic lipids, including PIPs, LPA, and LPS were eluted with chloroform/methanol/HCl/water (12:12:1:1) (3 × 1 mL).
- After addition of 1.5 mL of water and 0.113 mL of 2 M NaCl, the solution was vigorously shaken and centrifuged to collect the lower layer.
- After addition of 2 μL of 1 nmol/μL PIP2(8:0/8:0), each sample was dried under nitrogen gas.
- The sample was redissolved in 0.05 mL of methanol/70% ethylamine (100:0.065), followed by addition of 0.03 mL of 1 M ammonium bicarbonate.
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- 細胞を冷メタノール（2mL）に懸濁し、内標準 PIP1(16:0/16:0), PIP2(16:0/16:0), PIP3(16:0/16:0) 各2pmol/μLを50μL添加する。更に吸着を防御するためにPIP2(18:0/8:0) 1nmol/μLを2μL添加する。
- 2N塩酸1mLと水1mL 添加し、よく撹拌する。
- 2M 塩化ナトリウム水溶液 0.15mLとクロロホルム2mL を添加し、氷上で激しく撹拌する（３分）
- 遠心分離（2000g 5分）し、下層を分取する。
- 得られた抽出液にメタノール1.5mLを混和し、DEAEセルロースカラム(Wako Pure Chemicals)に注入する。
- クロロホルム／メタノール(1:1) 3 x 1mL、次いでクロロホルム／メタノール／28%アンモニア水／氷酢酸 (200:100:3:1) 3 x 1mLでカラムを洗浄する。
- 酸性脂質をクロロホルム／メタノール／濃塩酸／水 (12:12:1:1) 3 x 1mLで溶出する。
- 溶出液に水1.5mLと2M塩化ナトリウム水溶液 0.113mLを加え激しく撹拌し、遠心後下層を分取する。
- 1nmol/μL PIP2(8:0/8:0) 2μLを加え、溶媒を窒素気流で留去する。
- 得られた酸性脂質をメタノール/70%エチルアミン(100:0.065)0.05mLで再溶解後、1M炭酸水素アンモニウム水溶液0.03mLを混和し、試料とする。
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