Metabolomic analysis

1. The HL-60 cells were cultured in RPMI 1640 culture media supplemented with 10% FBS for two days. 
2. 1 × 10⁷ cells were seeded in 75 culture dishes overnight and treated with different concentration of IR-26 (0, 2.5, 5 10μM). 
3. After incubation for 8 hours, cells in different groups were harvest and washed with PBS for three times. 
4. After centrifuged, cells in EP tubes were kept in liquid nitrogen for 30s and stored under -80℃ condition. 
5. After thawing, all the samples were transferred into new tubes with 1000μL extracting solution (Methanal : Acetonitrile : H₂O = 2:2:1), then added steel ball.
6. Cells were grinded for 4min, and then ultrasonic for 5 min under ice cold conditions.
7. Repeated step 6 for twice.
8. Samples were left for 1 hour under -20℃.
9. Then, all the samples were centrifuged at 12000rpm for 15 min under 4℃, took 800μL supernatant from each sample into a new tube respectively.
10. Took 145μL of supernatant from each sample and mixed into two quality controls (800μL).
11. Concentrating all the samples with blowing nitrogen gas.
12. All the samples were then re-dissolved with 50μL of 80% Methanal, after centrifuged, the supernatants were used for metabolomic analysis. 
13. The targeted metabolomics experiment for central carbon metabolism was conducted by a commercially-available testing company, BIOTREE Biotech Co. Ltd. (Shanghai, China).  
14. Metabolite identification was based on TraceFinder search with home-built database containing about 300 compounds.

     Reagents: Methanal (CNW Technologies), Acetonitrile(CNW Technologies), Acetic acid (CNW Technologies), tributylamine (CNW Technologies)

    Apparatus: TSQ Quantiva (Thermo Fisher Scientific), centrifuge(Thermo Fisher Scientific)

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