Spectramax id3 microreader

A172 and U-373MG cells (1 × 10⁴ cells/well) were seeded in 96-well plates and incubated for 24 h to allow the cells to adhere to the wells. Both cell lines were treated with 0, 10, 25, 50, 100, and 200 μM luteolin and cultured for 24, 48, and 72 h. At the end of the set time, 10 µL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma, Louis, MO, USA) solution (5 mg/mL) was added to each well and incubated for 4 h at 37 °C. The medium (mixed with MTT solution) was removed from the wells, and 100 μL DMSO was added to each well and shaken for 10 min to dissolve the formazan crystals that formed in the cells. The absorbance of the dissolved formazan crystals was measured at 570 nm using a SpectraMax iD3 microreader (BioTek, Winooski, VT, USA). All data were derived from at least three independent experiments.

Cells were seeded on a six-well plate at a density of 2 × 10⁵ cells per well and incubated for 24 h to allow the cells to adhere and stabilize in the culture dish. Then, chrysophanol at a defined concentration was applied to the cells, cultured for 24 h, and then harvested. The total RNA was prepared using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA). The total RNA concentration was determined with a microplate spectrophotometer using the SpectraMax iD3 micro reader (BioTek, Winousk, VT, USA). cDNA was synthesized using a total RNA (2 μg) with a TOPscript^TM cDNA synthesis kit (Enzynomics, Dajeeon, Republic of Korea) by following the manual. Subsequently, quantitative real-time PCR was executed using the TOPreal^TM qPCR 2× PreMIX (SYBR Green with Low ROX) (Enzynomics, Dajeeon, Republic of Korea) with QuantStudio 1 (Applied Biosystems, Foster City, CA, USA). The relative mRNA levels were normalized using GAPDH as a housekeeping gene. The PCR primer sequences are shown in Table 1.

HaCaT, CAL27 and Ca9-22 (1 × 10⁴) cells were seeded in a 96-well plate and incubated in an incubator for 24 h to allow the cells to attach to the well surface. Both cell lines were subsequently treated with various polydatin concentrations and then cultured for 24–72 h. After the medium was removed, each well was treated with MTT solution and incubated at 37 °C for 4 h. Subsequently, the supernatant was removed, the formazan crystals were dissolved using DMSO, and the absorbance was measured at 570 nm using a SpectraMax iD3 microreader (BioTek, Winooski, VT, USA). Absorbance was measured in three independent experiments, and the results are expressed as mean ± standard deviation (SD).