Emax precision microplate reader

Cells were plated in 96-well plates at 4000 to 8000 cells per well and were allowed to adhere overnight. After 48 h culture with 5-FU, the IC₅₀ values were determined using a Cell Counting Kit-8 (DojinDo). We added 10 μl of CCK-8 solution to each well, incubated the plates for 3.5 h in an incubator at 37 °C with 5% CO₂, and then measured the optical densities at 450 nm using an Emax precision microplate reader (Molecular Devices).

Sil-15 cells were used to detect and quantify the transcriptional activity of retinoids added to the medium by monitoring β-galactosidase activity produced by the reporter cells. 96-well plates were coated with 0.1% gelatin for at least 2 h at 37 °C, washed twice with PBS, wrapped with parafilm and stored at 4 °C until use. Sil-15 cells were removed from stock culture flasks by trypsinization, counted and plated at 100,000 cells per well in the pre-coated 96-well plates. After attachment overnight in DMEM containing 10% FCS, the medium was replaced with fresh DMEM/10% FCS and serial dilutions of retinoid ligands, prepared in DMEM containing 10% FCS, were added at concentrations from 10^− 6 M to 10^− 14 M. The plates were incubated overnight at 37 °C / 5% CO₂. All concentrations for the ATRA standard curve and the other retinoid ligands were tested in triplicate. The next day, the assay plates were washed twice with PBS, fixed with 100 μl per well of 1% glutaraldehyde and 1 mM MgCl₂ in PBS for 15 min, washed twice with PBS and β-galactosidase activity detected by adding to each well 100 μl of freshly-prepared 0.2% X-Gal in 1 mM MgCl₂, 3.3 mM potassium ferricyanide and 3.3 mM potassium ferrocyanide in PBS. Plates were incubated for 6 h at 37 °C in 5% CO₂ and colour change at 650 nm measured on an Emax Precision Microplate Reader (Molecular Devices).

The fractionated ginger solutions based on BHI were prepared according to the broth microdilution method from 10% to 0.01%. Diluted samples (100 μL) were mixed with a suspension of each strain (100 μL). Before and after incubation with bacteria under each incubation condition, absorbance was measured at a wavelength of 600 nm using an ELISA microplate reader (EMax® Precision Microplate Reader; Molecular Devices, Sunnyvale, CA, USA). The lowest concentration with an absorbance gap smaller than 0.01 was determined as the MIC. To determine the MBC, 100 μL aliquots extracted from samples higher than the MIC were spread in BAP and incubated as described in the MIC test. The MBC was determined by confirming the lowest concentration without visible bacterial growth on the BAP.

MIC and MBC determinations were used to assess the antimicrobial efficacy of manuka oil against all bacterial strains. To determine the most potent antibacterial component, the agent (manuka oil) ranged from 10% to 0.019% using the broth microdilution method described in ISO 20776-1:2019 [18 ], and then the MIC was determined. A standardized suspension (100 μL) of each strain was added to 100 μL of the diluted agents. After incubation in a 5% CO₂ incubator at 37 °C for 24 h (for one week under anaerobic conditions for P. gingivalis), absorbance was measured using an enzyme-linked immunosorbent assay (ELISA) microplate reader (EMax^® Precision Microplate Reader; Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 600 nm. Then, the differences in absorbance values before and after incubation were compared. MIC was determined as the lowest concentration that inhibited bacterial growth, with an absorbance gap lower than 0.01 at 600 nm.
An aliquot (100 μL) was extracted in groups with concentrations higher than the MIC. To determine MBC, the aliquot was spread on the surface of BAP and subcultured. After incubation, including the MIC test, MBC was determined as the lowest concentration without visible bacterial growth on BAP.

The TGF-β bioassay (MFB-F11) developed by Tesseur et al.^{23 (link)} was used with embryonic fibroblasts from TGF-β1^–/– mice stably transfected with a TGF-β-responsive reporter plasmid containing a secreted embryonic alkaline phosphatase reporter gene (SBE-SEAP). MFB-F11 cells were grown in DMEM with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine and supplemented with 15 μg/ml Hygromycin B (Invitrogen), for 3 days. Cells were tested and found to be mycoplasma-free. Confluent cells were detached with trypsin, and resuspended in DMEM with 2.5% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine at a concentration of 4 × 10⁵ cells/ml. In 50 μl, 4 × 10⁴ cells were added to each well of a 96-well round-bottomed plate. Serial dilutions of test substances HES (in-house), Hp-TGM (in-house), and recombinant human TGF-β1 (R&D Systems) were then added to each well in a volume of 50 μl and incubated for 24 h at 37 °C. Subsequently, 20 μl of supernatant was aspirated from each well, added to an ELISA plate (NUNC) with 180 μl of reconstituted Sigma FastTM p-nitrophenyl phosphate substrate and incubated at RT in the dark for up to 4 h. Plates were read on at 405 nm on an Emax precision microplate reader (Molecular Devices).

The murine leukemic monocyte macrophage line, RAW 264.7, was obtained from Korea Cell Line Bank (KCLB No. 40071; Seoul, Korea) and grown at 37°C in a 5% CO₂ atmosphere in Roswell Park Memorial Institute medium (RPMI; Gibco, Carlsbad, CA, USA) 1640 containing 10% FBS (Gibco). The cells were then seeded at a concentration of 8.0 × 10⁴ cells/cm² in 12-well plates with 1 mL of media containing 2% FBS. Following 8 h culture, cells were incubated with 100 or 500 μg/mL of MFP for 0, 12, 24, or 48 h. After treatment with MFP, the cells were then incubated in FBS-free media with 0.5 mg/mL of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Life Technologies, Carlsbad, CA, USA) for 4 h. Formed crystals were dissolved in 1 mL of dimethyl sulfoxide (DMSO; Sigma, USA) and 100-μL aliquots were transferred to 96-well plates. The plates were read on an Emax Precision Microplate Reader (Molecular Devices, Sunnyvale, CA, USA), using a test wavelength of 590 nm and a reference wavelength of 620 nm. Cell numbers were calculated based on the standard curve generated from serially diluted cells. The effect of MFP on cell proliferation was expressed relative to the cell number at 0 h.