Materials and Reagents

1. 15-ml conical centrifuge tubes (Corning, Falcon^®)
   
2. Polypropylene microcentrifuge tubes of 1.5 ml (Eppendorf)
   
3. 0.3 ml polypropylene chromatographic vials (Focus Technology, model: VP91 ) with polypropylene cap and PTFE/silicone septa (Focus Technology, model: SC9291 )
   
4. 4 mm hydrophilic PTFE syringe filter with a 0.22 µm pore size (Nantong Filterbio Membrane, catalog number: FBS4PTFE022L )
   
5. 47 mm hydrophilic PTFE membrane with a 0.45 µm pore size (Nantong Filterbio Membrane, catalog number: FBM047PTFE045L )
   
6. Glass beads 212-300 µm (Sigma-Aldrich, catalog number: G9143-250G )
   
7. 1 ml disposable polypropylene syringe (B. Braun Medical, catalog number: 9166017V )
   
8. Millex-GS 33 mm sterile syringe filter with a 0.22 µm pore size (EMD Millipore, catalog number: SLGS033SS )
   
9. Milli-Q water
   
10. Liquid nitrogen
    
11. Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP) (Carl Roth, catalog number: HN95.2 )
    
12. Potassium dihydrogen phosphate (KH₂PO₄) (Carl Roth, catalog number: 3904.1 )
    
13. Orthophosphoric acid (H₃PO₄) (Sigma-Aldrich, catalog number: W290017 )
    
14. EDTA (Sigma-Aldrich, catalog number: EDS-100G )
    
15. Sodium chloride (NaCl) (Duchefa Biochemie, catalog number: S0520.1000 )
    
16. Potassium chloride (KCl) (Duchefa Biochemie, catalog number: P0515.1000 )
    
17. Di-sodium hydrogen phosphate (Na₂HPO₄) (Carl Roth, catalog number: P030.2 )
    
18. Magnesium chloride hexahydrate (MgCl₂·6H₂O) (Sigma-Aldrich, catalog number: M2393-100g )
    
19. Calcium chloride dihydrate (CaCl₂·2H₂O) (Duchefa Biochemie, catalog number: C0504.1000 )
    
20. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 258148-2.5L-D )
    
21. Sodium L-Asc (Sigma-Aldrich, catalog number: 4034-100G )
    
22. Acetonitrile HPLC gradient grade (VWR, HiPerSolv Chromanorm^®, catalog number: 83639.320 )
    
23. Phosphate-buffered saline (PBS) (see Recipes)
    
24. Extraction buffer (see Recipes)
    
25. HPLC mobile phase A (see Recipes)
    
26. HPLC mobile phase B (see Recipes)
    
27. Sodium L-Asc standard solutions (see Recipes)
    

Equipment

1. Scepter^TM handheld automated cell counter (EMD Millipore, catalog number: PHCC00000 )
   
2. Scepter^TM Sensors – 40 µm (EMD Millipore, catalog number: PHCC40050 )
   
3. Refrigerated table centrifuge and microcentrifuge
   
4. HPLC system (Shimadzu Scientific Instruments) equipped with:
1. LC pump (Shimadzu Scientific Instruments, model: LC-20AD )
   
2. Thermostated autosampler (Shimadzu Scientific Instruments, model: SIL-20AC )
   
3. Photo Diode Array detector (Shimadzu Scientific Instruments, model: SPD-M30A )
   
4. Column oven (Shimadzu Scientific Instruments, model: CTO-20AC )
   
5. Synergy hydro 4u Hydro – RP 80 Å 250 x 4.6 mm (Phenomenex, catalog number: 00G-4375-E0 ) directly mounted with a guard column AQ C18 4.0 x 3.0 mm (Phenomenex, catalog number: KJ0-4282 )

Software

1. LabSolution (Shimadzu Scientific Instruments)

Procedure