Butanol

Acetonitrile (CH₃CN), chloroform (CHCl₃), dichloromethane (CH₂Cl₂), butanol (C₄H₉OH), n-hexane (C₆H₁₄), formic acid and methanol (CH₃OH) were of analytical grade and were purchased from Merck (Darmstadt, Germany) and Sigma Aldrich (Seelze, Germany). Water (H₂O) was obtained using an 18 Ωm Milli-Q system (Millipore, Temecula, CA, USA).

For the analysis of oxidative lipid damage [67 (link)], the thiobarbituric acid reactive substances (TBARS) assay was employed. It is based on the detection of malondialdehyde (MDA), a marker for oxidative stress damage, which emits fluorescent light at 555 nm. Treated and untreated cells were washed once with PBS medium after 24 h metal ion mixtures treatment. A reagent containing 91.8 mM trichloroacetic acid (Merck, Darmstadt, Germany), 2.5 mM thiobarbituric acid (Merck, Darmstadt, Germany), 45.4 μM butylhydroxytoluene (Merck, Darmstadt, Germany) and 25 mM HCl (Merck, Darmstadt, Germany) was added to the pellet, which was thoroughly resuspended and homogenized twice (Bullet Blender Storm 24, Next Advance, Troy, New York, NY, USA) with 5 min of incubation on ice in between. The homogenate was incubated at 90 °C (Thermomixer R, Eppendorf, Hamburg, Germany) for 30 min followed by 10 min of incubation on ice. Butanol (Merck, Darmstadt, Germany) was added to the homogenate and centrifuged at 10,000× g for 10 min. Fluorescence (excitation/emission = 515/555 nm) and OD (650 nm) were measured on a Varioskan™ LUX (Thermofisher, Waltham, MA, USA) microplate reader. The results were expressed as F/OD relative to the untreated control.