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Incubated at 37 °C for 60 min, the mixture solution was used as the blank solution.
After 30 min, the mixture was diluted with water (100 mL).
After being homogenized for 2 min, the mixture was centrifuged (at 8,000 rpm, 10 min).
After 60 min, the mixture was centrifuged at 5000g for 15 min.
For the next 20 min, the mixture was stirred at 30 rpm.
After stirring for 1 min, the mixture was poured into a 60-mL Teflon-lined autoclave.
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About 1 g of fine powder was extracted with 4 ml of a hexane:diethyl ether (90 10 v/v) mixture using vortex for 1 min. The mixture was then centrifuged for 2 min at 20,238 g.
The mixture was incubated at 95°C for 5 min, followed by incubation at 50°C for 20 min. The mixture was then transferred to a 96-well filter plate.
An equal volume of chloroform was added to the homogenate followed by vortexing for 1 min. The mixture was centrifuged at 12,000 rpm for 10 min at 4°C and the upper phase was transferred to a new tube.
The serum mixture was brought to 20% saturation with ammonium sulfate solution under continuous stirring at 4°C for 30 min. The mixture was centrifuged at 3000 × g for 20 min, and the pellet was discarded.
Phase separation was performed by adding 0.2 ml of chloroform, vortexing for 15 sec and incubating at room temperature for 3 min. The mixture was then centrifuged at 12,000 g for 15 min at 4°C.
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