Cell Culture Preparation for 1D NMR

All samples should be kept on ice or at 4 oC during sample preparation or handling. Samples should be stored at -80 oC, but, ideally, samples should be immediately analyzed. In addition to keeping samples cold, there are four other issues that are critical to the successful preparation of metabolomics samples: (1) speed, (2) consistency, (3) random processing of samples, and (4) the efficient removal of all biomolecules and cell debris [8]. The processing of all metabolomics samples should proceed as quickly as possible while minimizing any loss in quality. Metabolites can chemically degrade or transform within milliseconds due to enzymatic activity, oxidation, chemical instability, or any number of other chemical processes [35]. Accordingly, rapidly inactivating and removing all biomolecules and cell debris (usually through methanol/ethanol precipitation) that may transform or bind a metabolite is a necessary step of the protocol (see Notes 9). Note: Keep methanol and water on ice throughout procedure!! Keep samples on ice as much as possible.

---

1. All media is collected. Media is spun at 300g for 5 min to pellet any derby or cells and transfered to new tube(s). 500uL of the media is reserved for metabolomic analysis. Save 1mL media separately for reserve analysis.

2. Cells are washed twice with 5 mL of PBS to remove debris. Discard the wash.

3. Lyse and quench cells with 1 mL of pre-chilled methanol at -20 oC. Incubate cells at -80 °C for 15 min.

4. Using a cell scraper, cell debris and methanol is detached and collected in a 2 mL microcentrifuge tube. Confirm cell detachment using a microscope and repeat lyse and quenching if necessary.

5. 2 mL microcentrifuge tube is centrifuged for 5 min at 15,000 g and 4 °C to pellet the cell debris.

6. Supernatant collected and transfered to a new 2 mL microcentrifuge tube.

7. Metabolome extraction is repeated by adding 0.5 mL of an 80%/20% mixture of methanol/water kept at -20°C to the cell pellet.

8. Centrifuge the cell pellet with the extraction solvent for 5 min at 15,000 g at 4 °C to pellet the cell debris.

9. Supernatant collected and transfered it to the 2 mL microcentrifuge tube containing the original methanol extract. Combine the two extraction supernatants into a single tube.

10. Metabolome extraction is repeated a third time by adding 0.5 mL of ice cold water to the cell pellet.

11. Cell pellet is centrifuged with the extraction solvent for 5 min at 15,000 g at 4 °C to pellet the cell debris.

12. Supernatant collected and transfered it to the 2 mL microcentrifuge tube containing the two previous extraction supernatants. Combine the three extraction supernatants into a single tube.

Note: If supernatant exceeds 1.5mL keep samples split and concentrate sample down in liophilizer and combining before either is fully dry (partially lyophile)

13. Sample split into two 2 mL Eppendorf tube. Aliquot 100 uL for MS analysis and the remainder of the sample is used for NMR analysis.

14. Pellet stored.

15. Samples are snap frozen in Liquid Nitrogen and freeze dried to remove methanol by speed vacuum centrifugation (SpeedVac R Plus, Savant) and water by lyophilization using FreeZone™ (Labconco, Kansas City, MO) for 24 hours and stored at -20°C until NMR analysis.