Please leave your comments or questions

Extraction of the Polar Metabolites from Adherent Mammalian Cells

Author: Li Quan, updated date: , view: 261, Q&A: 0
Tag: For IMM metabolomics plateform

Materials and Reagents

  1. Methanol
    MS Water
    Internal standard

  2. 1.5 uL tubes


  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.)

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.