Materials and Reagents

1. Cells to be assayed [in the development of this protocol, we used Plasmodium falciparum (3D7 strain) cell cultures and human red blood cells (kindly supplied by the Australian Red Cross)]
   
2. ¹³C-U-glucose (Cambridge Isotope Laboratories, catalog number: CLM1396 )
   Note: ¹³C-U-glucose refers to Universally-labelled glucose - i.e. where all six carbons are ¹³C-atoms.
   
3. Hexokinase (>130 Units/mg) (Sigma-Aldrich, catalog number: H4502 )
   
4. Adenosine triphosphate (ATP) (Sigma-Aldrich, catalog number: A6419 )
   
5. Magnesium chloride (MgCl₂, AnalaR) (VWR International, catalog number: 25108.260 )
   
6. Tris-HCl (pH 7.5) (Sigma-Aldrich, catalog number: T5941 )
   
7. Ammonium chloride (NH₄Cl) (Ajax, catalog number: 31-500G )
   
8. Nicotinamide adenine dinucleotide (NAD⁺) (Sigma-Aldrich, catalog number: N3014 )
   
9. NaHEPES (pH 7.4) (Sigma-Aldrich, catalog number: H3375 )
   
10. Ethylene glycol tetraacetic acid (Sigma-Aldrich, catalog number: E3889 )
    
11. Dithiothreitol (Sigma-Aldrich, catalog number: D0362 )
    
12. scyllo-Inositol (hereafter abbreviated to ‘sI’) (Sigma-Aldrich, catalog number: I8132 )
    
13. Glucose 6-phosphate (Sigma-Aldrich, catalog number: G7879 )
    
14. myo-Inositol 3-phosphate (Cayman Chemical Company, catalog number: CAY10007778 )
    
15. Chloroform (HPLC grade) (Thermo Fisher Scientific, catalog number: 10615492 )
    
16. Methanol (HPLC grade) (Thermo Fisher Scientific, catalog number: 10767665 )
    
17. Assay buffer (see Recipes)
    
18. Lysis buffer (see Recipes)
    
    Additional materials required for GC-MS
    
19. Methoxyamine hydrochloride (Sigma-Aldrich, catalog number: 226904-25G )
    
20. Pyridine (Sigma-Aldrich, catalog number: 270970 )
    
21. BSTFA + 1% TMCS (Sigma-Aldrich, Supelco, catalog number: 33148 )
    
22. Glucose (Sigma-Aldrich, catalog number: G8270 )
    

Equipment

1. 1.5 ml tubes (with safe-lock lids) (Eppendorf)
   
2. Bench-top centrifuge for 1.5 ml tubes
   
3. 1 ml, 200 µl, and 20 µl pipettes and accompanying tips
   
4. 37 °C water bath
   
5. Boiling water bath
   
6. Timer
   
7. Distilled (e.g. MilliQ) water supply
   
8. Light microscope, slides, and cover slips (for assessment of lysis)
   
9. Bench-top vortex or water bath sonicator (may be required for efficient lysis)
   
   Additional equipment required for GC-MS
   
10. Gas Chromatography-Mass Spectrometer (e.g. Agilent 7890B-5977A)
    
11. DB-5MS + DG column, (30m x 0.25 mm, with 10 m inert gap) (Agilent, J&W)
    
12. Ultra high purity helium
    
13. 1.5 ml tubes (Eppendorf)
    
14. 2 ml glass vials for mass spectrometry (e.g. Agilent, part number: 5182-0715 )
    
15. 9 mm caps with septa for glass vials (e.g. Agilent, part number: 5185-5820 )
    
16. 250 µl vial inserts (glass) (Agilent, part number: 5183-2085 )
    
17. Rotary vacuum concentrator (e.g. Christ, model: RVC 2-33 CD Plus )
    
18. Microdispensers and accompanying glass capillaries (50 µl adjustable, 200 µl) (Drummond Scientific Company)
    

Software

1. ChemStation software (MSD ChemStation D.01.02.16) (Agilent)

Procedure