Zeaxanthin

The following antioxidants were used in this study: Trolox (Vector Laboratories, CB-1000-2), Zeaxanthin (Santa Cruz, sc-205544), L-Sodium Pyruvate (Nacalai Tesque, 06977-34), α-tocopherol (Nacalai Tesque, 34114-54), Rutin (Nacalai Tesque, 30319-04), N-Acetyl-L-cysteine (Nacalai Tesque, 00512-84) and Ascorbic acid (Nacalai Tesque, 03420-52). Ascorbic acid (100 mM) was prepared in water and stored at −20 °C. Zeaxanthin (2 mM), α-tocopherol (10 mM), Rutin (10 mM) and NAC (100 mM) were prepared in DMSO and stored at −20 °C. Trolox (100 mM) and Sodium Pyruvate (100 mM) were stored at 4 °C. Each antioxidant was diluted in 500 µL of the culture medium at a final concentration before live cell imaging.

Carotenoids were extracted as described above and analyzed using high-performance liquid chromatography (1260 Infinity II, Agilent, CA, USA) equipped with a C30 column (YMC Carotenoid column, 250 mm, 5 μm pore size). Mobile phase A consisted of 15:81:4 Methyl tert-Butyl Ether (MTBE):methanol:water by volume, and mobile phase B consisted of 81:15:4MTBE: methanol:water by volume. Using a flow rate of 1.0 mL/min at 20 °C, a linear elution gradient from 100% A to 100% B over 15 min was followed by 12 min of 100% B before returning to mobile phase A over 3 min. HPLC standards (astaxanthin, lycopene, β-carotene, zeaxanthin, and canthaxanthin) were purchased from Santa Cruz Biotechnology for identification of carotenoid retention times. zeaxanthin was used to identify isozeaxanthin as this compound cannot be purchased, and these isomers are known to co-elute using C18 chromatography [20 (link)].