Fastprep 24 5g bead beating system

Heme was unbound and extracted from the bacterial hemoproteins using modified actone:HCl extraction methods [44 (link)]. The washed bacterial cell pellet was mixed with 1 mL of acetone:HCl (80:20) and 0.2 g of glass beads (212–300 µm), followed by homogenization for 30 s using a bead beater (FastPrep-24™ 5G bead beating system, MP Biomedicals, USA). After resting for 1 min, homogenization was repeated five more times. Subsequently, the lysate was incubated at − 20 °C for 20 min. Cell debris in lysate was removed by centrifugation (17,000×g, at 4 °C for 10 min), and the supernatant was used for measuring intracellular heme.
Heme amount was determined by measuring UV-absorbance using high-performance liquid chromatography (HPLC Agilent 1100) equipped with reverse-phase C18 column (C18, 3.5 μm, 150 mm × 4.6 mm, SunFire™). The temperature of the column oven was set at 40 °C. The mobile phase was composed of solvent A (methanol:acetonitrile = 10:90) and B (0.5% (v/v) trifluoroacetic acid in HPLC grade water). A linear gradient method (20–95%) of solvent A was applied for 0 to 7 min. The flow rate was maintained at 1 mL/min for a total analysis time of 10 min. The chromatogram peak of heme at about 6.7 min retention time was detected at a wavelength of 398 nm. The calibration curve was constructed using (0.01–1.5) µM hemin solution in acetone:HCl (80:20).

Zeaxanthin (5 mg/kg) was intravenously injected into the femoral vein of rats. The brain, liver, heart, lung, kidney, gut, spleen, muscle, adipose, and eyes were excised 90 min after the administration, rinsed with saline, and blotted dry with clean paper. Each weighed tissue sample was homogenized (FastPrep‑24™ 5 G bead beating system, MP Biomedicals, OH, USA) in four volumes of ice‑cold saline/methanol mixture (1:1, v/v). The tissue homogenate was then centrifuged at 9000 × g for 10 min, and 100 μL of supernatant was collected and processed using the same procedure as that used for the plasma samples.