Multiskan spectrum microplate reader

The cells were plated in 96-well plates at a density of 5 × 10⁴ cells per well. ATP-dependent luciferase luminescence was measured in cells incubated using the CellTiterGlo kit according to manufacturer’s instructions and analyzed using luciferase chemiluminescence Multiskan spectrum microplate reader (Thermo Electron Corporation, Waltham, MA, USA).

Human colorectal cells including Caco-2, HCT116, DLD-1 and HT29 cells seeded onto 96-well plates (5 × 10³ cells per well). After incubation for 24 h, shikonin derivatives (0, 6.25, 12.5, 25, 50 and 100 μg/mL) were added to the medium for additional 48 h. Then, cells were incubated with CCK-8 solution (10 μL, Dojindo Laboratories, Kumamoto, Japan) for 2 h. The absorbance was measured at 450 nm by Multiskan Spectrum Microplate Reader (Thermo Labsystems, Milan, Italy). The experiment was done in triplicate. The inhibitory rate of cell proliferation was calculated by the following formula:
$\text{Inhibitory}\hspace{0.17em}\text{rate}\hspace{0.17em}\left(\%\right)\hspace{0.17em}=1\hspace{0.17em}-\hspace{0.17em}\left({\text{OD}}_{\text{Sample}}-{\hspace{0.17em}\text{OD}}_{\text{Blank}}\right)/\left({\text{OD}}_{\text{Control}}-{\hspace{0.17em}\text{OD}}_{\text{Blank}}\right)\times 100.$

MTT and BrdU incorporation assays were performed to measure the proliferation of EpSCs as previously described [
13 (link),
14 ] . Optical density values were measured at 490 nm for the MTT assay and at 450 nm for the BrdU incorporation assay using the Multiskan Spectrum Microplate Reader (Thermo Fisher).

To examine the effect of JR on cell growth, EA.hy 926 cells were seeded into 96-well plates with 5 × 10³/well and cultured overnight. Then, cells were treated with varying concentrations of the JR (0.2, 0.4, 0.8, 1.6 mg/mL) for 24 or 48 h, and 20 μL MTT solution (5 mg/mL) was added to each well and incubated at 37°C for an additional 4 h. After carefully removing the culture medium, 150 μL dimethyl sulfoxide was added to each well to dissolve the crystals. A multiskan spectrum microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure absorbance of the converted dye in living cells at a wavelength of 570 nm.

The cell viability against the optimized formulation was assessed as described by Memvanga et al. [32 (link)]. In brief, Caco-2 cells were seeded on 96-well culture plates (2 × 10⁴ cells/well; 100 µL per well) and incubated in the culture media. After 24 h, the cells were washed with phosphate-buffered saline (37 °C) and treated with 100 µL of unloaded-SNEDDS or free Vox at various concentrations (from 0.3 to 4 mg/mL) diluted with Hank’s salt balanced solution (HBSS). After 2h of incubation, the cell were washed and treated with 100 µL of MTT solution (0.5 mg/mL in DMEM) and were incubated for 3 h (37 °C). To solubilize the formazan crystals formed during the incubation, 200 µL DMSO was added and the product of reaction was measured at 545 nm using a Multiskan Spectrum microplate reader (Thermo Fisher Scientific Inc.). The cell viability of the control cells (treated with HBSS) was defined as 100%.
The cell viability rates of the samples were calculated according to the following equation:
where A_s is the sample absorbance, and A_c is the absorbance measured after treating the cells with HBSS.

Optical rotations were recorded using MCP 200 Polarimeter (Anton Paar GmbH, Graz, Austria). Optical density (OD) values were read on a Multiskan Spectrum Microplate Reader (Thermo Scientific Inc., Shanghai, China). CD spectra were acquired on a Chirascan Spectrometer (Applied Photophysics Ltd., Surrey, UK). IR spectra were carried out on a Nicolet Nexus 670 spectrophotometer, in KBr discs. NMR spectra were obtained on a Bruker AVANCE 400 (Bruker Co. Ltd., Zurich, Switzerland). Thin-layer chromatography (TLC) was carried out on pre-coated silica gel GF-254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and column chromatography (CC) was performed over silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China, 200–300 mesh) on a Sephadex LH-20 (GE healthcare, Buckinghamshire, UK). Semi-preparative HPLC was performed on a Waters 1525 system using a semi-preparative Ultimate XB-C18 column (5 μm, 21.2 mm × 250 mm; Welch) coupled with a Waters 2998 photodiode array detector (Waters Corp., Milford, MA, USA). ESIMS data were measured on a Thermo LCQ DECA XP plus mass spectrometer (Thermo Scientific, Waltham, MA, USA). All reagents and solvents were of commercial quality.

Melanocytes were collected, washed three times with PBS, and subjected to cell counting. The cells were lysed using 0.2 mol/L NaOH (10⁶ cells/mL) and dispensed in a 96-well plate. The absorbance of the samples was measured at a wavelength of 475 nm [16 (link), 17 (link)] by using a Multiskan Spectrum microplate reader (Thermo Fisher Scientific). Each group was repeated three times.