*2.3. Measuring Uptake of Vitamin C*

Near-confluent cultures of HeLa cells in 60 mm dishes were incubated for 6 h (3 h + wash + 3 h, as above) with vitamin C at 50 or 200 μM. At the end of this incubation, or after a further 24 h

incubation in fresh medium without vitamin C, cells were washed with PBS and scraped with a silicone rubber scraper into suspension in PBS. A cell count was carried out. The suspension was centrifuged (400× *g*, 5 min) and the pellet resuspended in 250 μL of PBS to which an equal volume of 10% metaphosphoric acid was added before storing at −20 °C.

The frozen, acidified samples were thawed, and centrifuged (3500× *g* at 4 °C for 10 min). A volume of 100 μL clear supernatant was mixed with 400 μL of the mobile phase (2% acetonitrile in 2.5 mM NaH2PO4, 2.5 mM dodecyltrimethyl ammonium chloride and 1.25 mM Na2EDTA in Milli-Q water) for direct determination of vitamin C by high-performance liquid chromatography (HPLC). For separation of vitamin C from interfering sample constituents, a Chromolith Performance RP18-e, 4.6 mm × 100 mm column was used, with a Chromolith Performance RP18-e, 4.6 mm × 10 mm guard column (Phenomenex, Torrance, USA). The injection volume was 5 μL and the flow rate was 6.0 mL/min. A variable wavelength ultraviolet (UV) detector was used at 264 nm.
