| S Preparation
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HEK293 cells were maintained in DMEM (Niss … HEK293 cells were maintained in DMEM (Nissui Pharmaceutical) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin (Sigma-Aldrich), and 100 μg/ml streptomycin (Gibco) at 37°C in the presence of 5% CO2 gas.
HEK293 cells (1.0 × 105 cells) were incubated with recombinant PS-PLA1 in 100 μl Opti-MEM in the presence or absence of 0.1% BSA at 37°C for 1 h. The cultures were centrifuged at 1,000 g for 3 min. The supernatants and cell pellets were deproteinized with 160 μl and 200 μl acidic methanol (pH 4.0), respectively, 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 20 μl of the filtrate was subjected to LC-MS/MS. of the filtrate was subjected to LC-MS/MS.
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