El808 microplate reader

Mice were fed a high fat diet (HFD) consisting of 60% fat, 20% carbohydrate, and 20% protein (total 5.2 kcal/g, D12492 60% kcal diet, Research Diets Inc, NJ) as described previously [19 (link)]. Mice were placed on the HFD diet at 6 weeks, and kept on the diet for 20 weeks with serial weights and the presence of insulin resistance monitored by metabolic analyses.
At 22 weeks, mice were fasted overnight (16 h) with free access to water before injecting intraperitoneally with D-glucose (2 g/kg). Blood was obtained by tail nick at baseline, 30, 60, and 120 min after glucose administration [19 (link)]. The animal was briefly anesthetized by isoflurane for 3–5 min, whole blood obtained from the intraorbital retrobulbar plexus was allowed to clot at RT for 30 min before centrifugation at 4°C to separate serum from clotted blood component, and serum was stored at -80°C until insulin was measured using Ultra Sensitive Mouse Insulin ELISA Kit (Crystal Chem Inc., Downers Grove, IL). Absorbance was read at 450 nm in an EL808 microplate reader (BIO-TEK Inc., Winooski, VT) linked with the KC Junior program.

MTT assay was performed according to Mosman with minor modifications [36 (link)]. Briefly, an MTT solution (0.5 mg/mL final concentration) was added to the cells, which were incubated at 37 °C for 1 h in the case of the ADF cells and BLEC, or 30 min in the case of HLEC. Then, for the solubilization of formazan crystals, an equal volume of a solution of isopropanol containing 0.4 N HCl was added to the cell medium. Cells were maintained in agitation for 10 min at 37 °C. The absorbance of the solutions was then read at 563 nm with a EL-808 microplate reader (BioTek Instruments, Winooski, VT, USA).

PC-3 (1 × 10⁴ cells) and DU145 (1.1 × 10⁴ cells), RWPE-1 and PWR-1E (1.2 × 10⁴ cells) were seeded into flat-bottomed 96-well plates in 100 µL media per well. After the incubation period (40 h for cancer cells and 72 h for normal cells), cells were treated with siEphA2 (50 nM) alone and in combination with JIB-04 (260 nM) in 100 µL fresh antibiotic-free medium for 48 h. At the end of the treatment period, the media was removed and a mixture of 10 µL WST-8 reagent (Dojindo) with 100 µL fresh medium was added to each well. After 4 h incubation at 37 °C followed by agitation for 1 min, absorbance was measured at 450 nm and with 650 nm set as a reference wavelength (Versa max microplate reader, Molecular Devices or EL808 microplate reader, BioTek). After subtracting the absorbance of blank (only medium), the net absorbance value (A₄₅₀−A₆₅₀−A_blank) was normalized to UT control value and graphed as percentage cell viability. The half-lethal concentration (LC₅₀) value of JIB-04 was calculated by CompuSyn software [34 ].

RAW264.7 macrophages were seeded at a density of 1 × 10⁴ cells/well in 96-well plates in complete DMEM and allowed to attach overnight in humidified 5% CO₂ incubator at 37 °C. Twenty-five thousand THP-1 cells per well were seeded in 96-well plates with complete RPMI-1640 medium and 162 nM of PMA for monocyte differentiation into macrophage-like cells. Cells were incubated at 37 °C with 5% CO₂ for 48 h and for an additional time of 24 h, in which time the spent medium was replaced with a complete RPMI-1640 medium. RAW264.7 and THP-1 macrophages were treated with undigested and digested samples at 0.5 mg/mL of complete medium for 24 h. After incubation, the spent medium was replaced with 100 µL of serum-free medium with CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay solution (ratio 9:1, v/v) for 2 h at 37 °C in a humidified incubator with 5% CO₂. Absorbance was read at 490 nm on an EL808 microplate reader (Biotek Instruments, Winooski, VT, USA). Untreated RAW264.7 and THP-1 cells were considered as negative control. Cells stimulated with LPS (Sigma-Aldrich Co., St. Louis, MO, USA) were included as a positive control. Cell viability was expressed as a percentage of untreated cells (negative control, C−). Data represent the mean and the standard deviation of eight biological replicates.

To ensure that OMV production was not associated with impaired growth phenotypes, growth curves of the WT and ΔtolA strain were performed. Bacterial cells were inoculated into LB broth from frozen cryostocks and grown overnight. The resulting culture was standardized to 1 OD, and diluted 1/100 in LB into flat-bottom 96-well NUNC microtiter plates (Thermo Fisher Scientific, United States). Strains were grown for 24 h (h) in LB media at 37°C with continuous shaking, where OD_{600 nm} was measured every 2 h in a BioTek EL808 microplate reader (BioTek, Winooski, VT, United States). Growth of each strain was measured in triplicate from 6 biological replicates (n = 6) and Mann–Whitney U tests were performed to determine OD values that differed significantly (p < 0.05) between WT and ΔtolA at all time points.

The CellTiter 96® AQ_ueous One Solution cell proliferation assay (MTS, Promega) was used to examine cell viability. Cells were seeded into 96-well plates at 5 × 10³ cells/well. They were pretreated with U0126 at 10 μM (Cell Signaling; 9903) and then treated with escalating doses of pirfenidone or with 0.5 mg/mL of pirfenidone and 10 μM of cisplatin for 72 h. After the treatment period, 20 μL of the MTS solution was added and incubated at 37 °C for 1 h. Plates were read at 490 nm in a BioTek EL808 microplate reader. Treatments were compared to their vehicle control.