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Extraction of the Polar Metabolites from Adherent Mammalian Cells
Author: Li Quan, updated date: , view: 17, Q&A: 0
Tag: For IMM metabolomics plateform

Materials and Reagents

  1. Methanol
    Acetonitrile
    MS Water
    Internal standard

  2. 1.5 uL tubes


Procedure

  1. 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)

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

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

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

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

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

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

  8. 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.

  9. Dry the supernatants under a stream of N2 gas. 

  10. 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.)


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