Stable Isotope Resolved Metabolomics Studies in ex vivo TIssue Slices

Materials and Reagents

1. 25 ml T Flasks NC vent cap (SARSTEDT AG & Co, catalog number: 83.1810.002 )
   
2. Portable container for liquid nitrogen (Nalgene plastic dewar) (Thermo Fisher Scientific, catalog number: S34074C )
   
3. Sterile syringes and needles (Thermo Fisher Scientific, catalog number: 10142534 )
   
4. Disposable transfer pipets (Samco fine tip, 1 ml) (VWR International, catalog number: 16001192 )
   
5. Aerosol barrier tips for 1 ml and 1-200 μl (Thermo Fisher Scientific, catalog number: 02-707-42 )
   
6. Screw cap plastic vials (2 ml) color coded caps (yellow, blue, green and red) (USA Scientific, catalog number: 1420-8706 , 1420-8701 , 1420-8702 and 1420-9704 )
   
7. Snap top plastic vials (1.5 ml) (USA Scientific, catalog number: 1615-5510 )
   
8. 15 ml Falcon tubes (SARSTEDT AG & Co, catalog number: 62.554.205 )
   
9. Dialyzed, sterile filtered fetal bovine serum (FBS) (free of serum metabolites),10-12 kDa (Atlanta Biochemical, catalog number: S12650 )
   
10. Tracer examples: ¹³C₆-glucose, ¹³C₂-1, 2-glucose, ¹³C₅,¹⁵N₂-glutamine
    
11. Sources: ¹³C₆-glucose/D-glucose ([U-¹³C], 99%) (Cambridge Isotope Laboratories, catalog number: CLM-1396-CTM), ¹³C₂-1, 2 glucose/D-glucose (1, 2-¹³C₂, 99%) (Cambridge Isotope Laboratories, catalog number: CLM-504), ¹³C₅, ¹⁵N₂-glutamine/ L-glutamine (¹³C₅, 99%; ¹⁵N₂, 99%) (Cambridge Isotope Laboratories, catalog number: CNLM-1275 ) or
    Isotec: D-¹³C₆-glucose (Sigma-Aldrich, catalog number: 660663 ), ¹³C₂-1, 2 glucose (Sigma-Aldrich, catalog number: 661422 ), L-Glutamine-¹³C₅, ¹⁵N₂ (Sigma-Aldrich, catalog number: 607983 )
    
12. Penicillin + Streptomycin: GE Healthcare PEN/STREP/FUNGIZONE 100 ml (Thermo Fisher Scientific, catalog number: SV3007901 )
    
13. Protocol^TM 10% Neutral buffered formalin (Thermo Fisher Scientific, catalog number: 032-059 )
    
14. 25% (w/v) sterile filtered ¹³C glucose (0.2 μm) in PBS (Stock solution can be frozen, aliquoted, and stored at 4 °C)
    
15. Liquid nitrogen
    
16. 70% ethanol (v/v)
    
17. 60% acetonitrile in water (v/v) (Sigma-Aldrich, catalog number: L010400 )
    
18. Sodium chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-1 )
    
19. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
    
20. Sodium phosphate dibasic (Na₂HPO₄) (Sigma-Aldrich, catalog number: S0876 )
    
21. Potassium phosphate monobasic (KH₂PO₄) (Sigma-Aldrich, catalog number: P9791 )
    
22. Amino acids: Glycine, L-Arginine, L-Asparagine, L-Aspartic acid, L-Cystine 2HCl, L-Glutamic Acid, L-Glutamine, L-Histidine, L-Hydroxyproline, L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine disodium salt dehydrate, L-Valine
    
23. Vitamins: Biotin, Choline chloride, D-Calcium pantothenate, Folic Acid, i-Inositol, Niacinamide, Para-Aminobenzoic Acid, Pyridoxine hydrochloride, Riboflavin, Thiamine hydrochloride, Vitamin B12
    
24. Calcium nitrate [Ca(NO₃)₂^.4H₂O]
    
25. Magnesium sulfate (MgSO₄)
    
26. Glutathione (reduced)
    
27. Phenol Red
    
28. Relevant medium (e.g. DMEM, RPMI, other defined medium) which lacks the tracer of interest:
    a.Dulbecco′s Modified Eagle′s Medium (DMEM) is a powder formula, free of glucose, glutamine, pyruvate bicarbonate, and phenol red, giving considerable flexibility in formulation for SIRM studies (Sigma-Aldrich, catalog number: D5030 ) (see Recipes)
    b.RPMI 1640 is a liquid medium free of glucose and glutamine, but contains bicarbonate and phenol red (MP Biomedicals, catalog number: 091646854 ) (see Recipes)
    
29. 0.2 µm sterile filtered Phosphate Buffered Saline (PBS) (see Recipes)
    
30. Medium composition for 0.2% ¹³C₆-glucose, 2 mM ¹²C-Gln (100 ml) (see Recipes)
    
31. Medium composition for 0.2% ¹²C glucose, 2 mM ¹³C₅, ¹⁵N₂-Gln (100 ml) (see Recipes)

Equipment

1. Class II Biosafety Hood
   
2. Trigas incubator with oxygen sensor and CO₂ sensor (Thermo Fisher Scientific, model: Hera cell 150i )
   
3. Sterilized rocker (Rotoshake Genie) (Scientific Industries, model: SI-1100 )
   
4. Liquid nitrogen freezer for storage
   
5. K₂-EDTA vacutainers (“purple top”) (BD, catalog number: 366643 )
   
6. Refrigerated centrifuge with swing out rotor that can accept vacutainers [e.g. Sorvall Legend X1R (Thermo Fisher Scientific, catalog number: 75-004-261 ) with a rotor (Thermo Fisher Scientific, catalog number: 75003181 )]
   
7. Pipettors (variable size ranges) (USA Scientific ErgoOne)
   
8. Weck Knife/Dermatome (George Tiemann & Co., catalog number: 222-5-523 )
   
9. Weigh boats (Thermo Fisher Scientific, catalog number: 08732113 and 08732115 )
   
10. 4-place balance (Thermo Fisher Scientific, Mettler-Toledo, catalog number: 0133525 )
    
11. Ice bucket (Thermo Fisher Scientific, catalog number: 02-591-44 )
    
12. Sharp dissecting scissors (Thermo Fisher Scientific, catalog number: 08940 )
    
13. Excelta™ Plastic Tweezers (Thermo Fisher Scientific, catalog number: 17-456-066 )
    
14. Digital camera

Procedure