Hdtma

The CAS-Fe³⁺ solution was prepared as described by Schwyn and Neilands with minor modifications.^{[35 (link),38 (link)]} All glassware was rinsed with 6 M hydrochloric acid, followed by double distilled water prior to use. In a graduated glass cylinder 6 mL of a 10 mM solution of hexadecyltrimethylammonium bromide (HDTMA, Oakwood Chemical) was diluted to approx. 40 mL with double distilled water. While stirring, 1.5 mL of a ferric iron solution (1 mM iron(III) chloride hexahydrate (Sigma–Aldrich) in 10 mM hydrochloric acid) was added. Upon slow addition of 7.5 mL of 2 mM aqueous Chrome Azurol S (dark orange, ThermoFisher Scientific) the solution adopted an intense blue coloration. In a separate glass flask, 9.71 g piperazine-hexahydrate (Sigma–Aldrich) were dissolved in double distilled water (ca. 20 mL) and cooled on ice. 6.25 mL of 12 N hydrochloric acid were carefully added and this buffered solution was poured into the glass cylinder containing the CAS-Fe³⁺ solution before double distilled water was added to a final volume of 100 mL. Finally, 5-sulfosalicylic acid (Sigma–Aldrich) was added to a final concentration of 4 mM and the solution was stored in a light-protected polyethylene bottle at room temperature. This CAS-Fe³⁺ solution was used for all in vitro experiments over the course of several weeks.