*3.1. Ascorbate in Cell Culture Media*

Phosphate Buffered Saline (PBS) solution was prepared from PBS tabs (Sigma Aldrich, St. Louis, MO, USA) dissolved in double deionized, filter sterilized water. Hyclone RPMI 1640 (Thermo Fisher Scientific, Waltam, MA, USA) was either used alone or after overnight chelation with Chelex 100 resin and the addition of 1 mM diethylenetriaminepentaacetic acid (DTPA). PBS or media was placed in 6-well culture dishes in a cell culture incubator (5%/95%: CO2/air; 37 °C) or at room temperature. Low trace metal ascorbic acid (Sigma Aldrich, St. Louis, MO, USA) was dissolved at a concentration of 10 mM in deionized water before addition to culture dishes. At specified times, aliquots were removed and acidified in 15% perchloric acid (PCA) containing

5 mM DTPA for vitamin C analysis as described below. Cell culture media containing 10% FBS from different commercial sources (Hyclone, Thermo Fisher, Sigma or ATCC) or aliquots of FBS alone were also acidified with an equal amount of PCA, as described above for the analysis of ascorbate content. However, no ascorbate was detected unless ascorbate was added to these solutions.
