Acidic phospholipids （PA, LPA, PS, LPS, PI）

Cultured cells (3 to 6 × 106 per 100 mm plate) were scraped with 2 mL of ice-cold butanol, and 50 pmol of LPA(17:0) and PA(14:0/14:0) were added as an internal standard.
2 mL of water was added to each suspension, followed by vigorous shaking for 3 min while cooling with ice.
Samples were centrifuged at 1000g for 5 min, and the upper layer was collected.
After the addition of 1 mL of methanol and 1 mL of chloroform, each sample was applied to a DEAE-cellulose column.
Column-bound lipids were washed with chloroform (1 mL) and chloroform/methanol (1:1) (3 × 1 mL) in series.
Acidic lipids, containing PA, PS, and PI, and their lyso forms, were eluted with chloroform/methanol/28% aqueous ammonia/acetic acid (200:100:10:6.7) (3× 1 mL).
After removing organic solvent from each eluate under nitrogen gas, 0.05 mL of water and 0.1 mL of 2-propanol were added to about 0.05 mL of residue.

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