Extraction of the Polar Metabolites from Adherent Mammalian Cells

Methanol
Acetonitrile
MS Water
Internal standard

1.5 uL tubes

Methanol-water (80:20, v/v) or Methanol-Acetonitril-water (40:40:20) with appropriate concentration of the internal standard stored at -80 ℃, as the quenching solution (The concentration of the standard needs to be optimized and may be used in a range from 0.1 to 100 ng/ul of quenching mix)

Wash the plate with PBS or Tris-HCl (pH=7.2) or milliQ water for 3 times

Added 200-1000 µl of the quenching solution which was sufficient to cover the surface of seeded cells. 

Immediately place the culture dishes in -75 °C for 10 min to allow for complete metabolic quenching.

The cells are then scraped off the culture dish on ice, and transferred into a 1.5 mL tube

Vortex for 30 s and ultrasonic on ice for 2 min, and then freeze at -20 ℃ for 10 min. repeated 3 times.

Centrifuged at 12000 rpm for 10 min at 4 °C.

Collect supernatant and add a 200 µl of quenching mix to the pellet and vortex hard. Re-spin the tube and collect the supernatant. Repeat step.
Pool the three supernatants obtained.

Dry the supernatants under a stream of N2 gas. 

Redissolved the dried extract in MS grade Acetonitrile(5% 100 mM ammonium acetate
(redissolve volume is dependent on cell number and MS-sensitivity range. For 5 million THP1 cells used 180 µl volume, etc.)