Isopropyl alcohol

We used FFAs at a concentration of 0.5 mM (OA/palmitate, 1:1 equimolar mixture) to induce fat-overloading in cells [27 (link)]. After exposure to BPA or BPA plus FFAs for 24 h, cells were washed twice with PBS, fixed with 3.7% formaldehyde in PBS for 30 min, and washed twice with PBS. Intracellular TGs were stained with 0.35% Oil Red O powder (Sigma-Aldrich) in isopropyl alcohol (Sigma-Aldrich) for 30 min. Excess stain was removed by washing with 70% isopropyl alcohol and PBS. The stained lipid droplets were dissolved in isopropyl alcohol containing 4% Nonidet P-40 (Sigma-Aldrich) and quantified at 510 nm on a Synergy HT microplate reader (Biotek, Winooski, VT).
Intracellular lipid content was measured using an AdipoRed assay (Lonza, Allendale, NJ) according to the manufacturer’s instructions. Cells were incubated with 3 mM NAC for 1 h before BPA exposure for 24 h. The stained cells were washed with water, and observed under a fluorescence microscope (Leica DMi8, Leica Biosystems, Wetzlar, Germany). Images were recorded for five different fields of observation and analyzed using ImageJ 1.43u (National Institutes of Health, Bethesda, MD, USA).

The acid index of fuel samples taken from the bottom of tanks at the SE location was measured by acid titration using the ASTM standard D974 (D27 Committee, 2008 ) method at the 3, 7, and 9 months time points. Approximately 20 g of B20 suspended in 100 mL of titration solvent (100:1: 99 Toluene/Water/Isopropyl alcohol) and 0.5 mL of an indicator solution was titrated using a 0.1 N solution of KOH dissolved in Isopropyl alcohol (Sigma Aldrich).

Once positive, a bacterial pellet was obtained by successive centrifugation steps [37 (link)]. Briefly, 1.4 mL of bottle solution was transferred to an eppendorf tube, which was centrifuged at 1450 rpm (200× g) for 5 min. The supernatant was collected and centrifuged at 12,210 rpm (14,170× g) for 1 min, and the pellet was washed with distilled water (900 µL), vortexed for 30–60 s, and centrifuged at 13,000 rpm (16,060× g) for 2 min. The bacterial pellet was suspended in 300 μL of distilled water and vortexed for 30 s at room temperature. Then, 900 μL of absolute ethanol (Dinamica, São Paulo, Brazil) was added, vortexed for 30–60 s, and centrifuged at 13,000 rpm (16,060× g) for 2 min. The supernatant was discarded, and the pellet was re-suspended in 100 µL of extraction solvent (formic acid—isopropyl alcohol—water, 17:33:50 v/v) (formic acid and isopropyl alcohol from Sigma-Aldrich, Missouri, EUA). The suspension was vortexed for 30–60 s and centrifuged for 2 min at 13,000 rpm (16,060× g). The clean supernatant extract was kept at room temperature for further analysis [39 (link)].