Multilabel counter

Macrophages were equilibrated with NBD cholesterol (1 µg/mL) for 12 hours. These cells were washed with PBS and incubated with oxLDL (50 µg/mL) in RPMI 1640 medium for another 12 hours in the presence of apoAI (10 µg/mL) or HDL (50 µg/mL). The fluorescence‐labelled cholesterol released from the cells into the medium was analysed using a multilabel counter (PerkinElmer) with 485 nm excitation and 535 nm emission.

HUVECs were trypsinized and seeded at 10⁴ cells/well, in 96-well gelatin coated plates. After 24 h, complete medium was removed and renewed with hormone-free low serum (0.5% CS-FBS) medium. Samples were then incubated for 24 h, to starve HUVECs and achieve a quiescent state. Following these pre-incubations, different concentrations (12.5 μM–100 μM) of formononetin medium were replaced. Cells receiving DMSO (0.1%) served as vehicle controls, and were equivalent to no treatment. After 24 or 48 h, cell proliferation was assessed by XTT for 4 h. The spectrophotometrical absorbance of each well was measured by a Multilabel counter (Perkin Elmer, Singapore). The wavelength used to measure absorbance of the formazan product was 490 nm. The reference wavelength was 690 nm. Cell viability data were expressed as a percentage of calculated cell viability.

Nitric oxide in the culture medium is stably present as nitrite (NO₂-) which can be assayed by using the Griess reagent (Madison, WI, USA). According to the manufacturer's manual, RAW264.7 cells (1.0 × 10⁶ cells) were seeded into each well of 12-well plates. After preculture for 24 h, cells were starved by being cultured in serum-free medium for another 2.5 h to eliminate the influence of FBS. The cells were then treated with or without crocetin for 1.5 h before exposure to 40 ng/ml LPS for 12 h. The level of nitric oxide released into the culture medium was determined by measuring absorbance at 530 nm in a microplate reader.
The intracellular reactive oxygen species (ROS) generation of cells was investigated using DCFH-DA as a well-established compound to detect and quantify intracellular-produced hydrogen peroxide [45 (link)]. In brief, RAW264.7 cells were plated in a 96-well plate and pretreated with crocetin for 2 h before exposure to LPS (40 ng/ml) for 12 h. DCFH-DA was added to the medium with a final concentration of 20 μM for an additional 30 min. The fluorescence intensity was then measured at an excitation (485 nm) and emission (530 nm) wavelength using a fluorescent Multilabel Counter (Perkin-Elmer) and was expressed as the percentage of DMSO vehicle control in the absence of LPS. Data are the mean ± SD of three separate experiments.

The LAB strains were grown in MRS broth at 35 °C. After 12 h, the cultures were centrifugated (8000 rpm for 15 min at 4 °C). Subsequently, the bacterial cells were washed three times with physiological solution (NaCl 0.9%) and resuspended in the same solution to an optical density of 0.5 on the MacFarland scale (OD₅₈₀), to standardize the bacterial density at 10⁸ CFU/mL. The OD₅₈₀ of the BS was measured using a spectrophotometer (Multilabel Counter—PerkinElmer 1420, San Jose, CA, USA). The subsequent tests were conducted in triplicate and the measurements were carried out in duplicate.

The membrane-permeable probes HE and DCFH-DA were used to assess levels of ROS using methods that have been described previously (Liu et al., 2005 (link)). Oxidation of HE by ROS, preferentially superoxide, forms red fluorescent ethidium (ETH) (Benov et al., 1998 (link)), whereas oxidation of DCFH-DA by ROS, particularly hydrogen peroxide, yields fluorescent 2,7-dichlorofluorescein (DCF) (Myhre et al., 2003 (link)). For the purpose of these experiments, HBECs were incubated in culture medium containing 10 μM HE or 20 μM DCFH-DA at 37°C for 30 min. Then, cell medium was replaced with fresh medium. After stimulation with CSE for 30 min, cells were washed and detached with trypsin/EDTA, and the fluorescence intensity of the cells was analyzed by use of a multilabel counter (PerkinElmer, Waltham, MA) at 518 nm excitation and 605 nm emission for ETH, and at 488 nm excitation and 530 nm emission for DCF. Images of the cells were also obtained by examining them using a Nikon TE2000-U florescence microscope (Tokyo, Japan).

Interaction of proteins was determined using a modification of the Checkmate mammalian two-hybrid system (Promega). Two vectors were used, pAct and pBind3-D. pAct (Promega) contains the herpes simplex virus VP16 activation domain followed by a multiple cloning site. pBind3-D [64 (link)] is a modification of pBind (Promega) in which the DNA-binding domain of the yeast GAL4 gene followed by an altered multiple cloning site and the vector lacks the Renilla luciferase module.
N2a cells were seeded on 96-well plates and transfected in parallel with pAct, pBind3-D, peGFP (Clontech), and a pG5luc (Promega) expressing firefly luciferase under control of GAL4 [64 (link)]. The next day fluorescence generated by eGFP was determined for normalization and light emission generated by luciferase activity was detected after adding Bright-Glo (Luciferase Assay System, Promega) with a Multilabel Counter (PerkinElmer). Luciferase activity was normalized to eGFP-fluorescence. All transfections and analysis were performed in septuplicate and experiments repeated three times. Average relative luciferase light units and S.D. were determined using the Prism software.