Arium pro ultrapure water system

HPLC-grade ethanol (EtOH) and dichloromethane were purchased from Merck (Merck Co., Darmstadt, Germany). The LC-MS-grade solvents acetonitrile (ACN), methanol (MeOH) and formic acid (FA) were purchased from Fisher Chemical (New Jersey, USA). Chemical standards of lutein, β-carotene, zeaxanthin, and lycopene were purchased from Sigma (St Louis, MO, USA); those of β-cryptoxanthin, all-trans-canthaxanthin, all-trans-fucoxanthin, all-trans-astaxanthin, violaxanthin and neoxanthin were obtained from ChromaDex (Santa Ana, CA). All other reagents and solvents were of high analytical grade unless otherwise stated. Water was obtained from arium® pro Ultrapure Water Systems (Sartorius, Goettingen, Germany). All solvents were degassed before use.

25‐mm borosilicate glass coverslips (VWR International, Radnor, PA, USA) were passivated with poly‐L‐lysine‐grafted polyethylene glycol (PLL‐PEG) to minimize unspecific interactions with the glass surface and partly functionalized for cell adhesion with a peptide (CGRGDS) containing the motif arginyl‐glycyl‐aspartic acid (RGD) 28. PLL‐PEG‐RGD was a kind gift of Jacob Piehler (Osnabrück University). Coverslips were sonicated (Sonorex SUPER RK 102H, Bandelin, Berlin, Germany) in isopropyl alcohol (Sigma‐Aldrich, St. Louis, MO, USA) for 20 min at 23 °C, rinsed with bidistilled water (Arium Pro Ultrapure Water Systems, Sartorius, Goettingen, Germany), dried with nitrogen, and plasma cleaned for 10 min at 80% power and 0.2–0.3 mbar (Zepto B; Diener Electronic GmbH, Ebhausen, Germany). Ten microlitre of 0.8 mg·mL⁻¹ PLL‐PEG‐RGD solution in 1 × phosphate‐buffered saline (PBS) pH 7.4 (prepared from 10 × concentrate, catalog# D1408; Sigma‐Aldrich) was incubated between two coverslips for approximately 90 min at 23 °C. Coverslips were separated by rinsing with bidistilled water, dried with nitrogen, and stored at −20 °C until further use in six‐well plates (Greiner Bio‐One International GmbH, Frickenhausen, Germany) covered with argon.