Fluostar optima spectrophotometer

HUVECs (2 × 10⁴ cells/well) were allowed to attach to 96-well plates for 4 h in MV2 medium containing 5% (v/v) MV2 Supplement Mix and subsequently incubated in the same medium with DMSO (vehicle) or SGLT2 inhibitors for 24 h. CellTiter 96® AQ_ueous One Solution Aqueous Cell solution was added 1.5 h prior to the end of incubation, at which point A₄₈₅ was measured on a BMG Labtech FLUOstar Optima spectrophotometer microplate reader.

HUVECs (2 × 10⁴ cells/well) were allowed to attach to 96-well plates for 4 h in MV2 medium containing 2.5% or 5% (v/v) MV2 Supplement Mix (Promocell C-39226) containing serum and growth factors and subsequently incubated in the same medium with DMSO (vehicle) or SGLT2 inhibitors for 12 h. BrdU reagent was then added and cells further incubated for 24 h. BrdU incorporation was assessed by measuring A_485/520 on a BMG Labtech FLUOstar Optima spectrophotometer microplate reader.

Group III with the ratio of 40:60% with an emulsifier (5%) and group IV with the ratio of 40:60% with 2.5% emulsifier was evaluated for the monitoring of stability in a room (24 °C ± 1) and fridge (2 °C ± 1) temperatures.
The 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH) is a stable radical with a maximum absorbance at 517 nm that can readily undergo reduction by an antioxidant. The scavenging effect on DPPH radical was determined by the method reported earlier with minor modifications [13 (link)]. Different concentrations of grape seed oil in methanol (20 μl) were mixed with 270 μl of 0.004% methanolic solution of DPPH. The mixture was shaken vigorously and left to stand for 30 min in dark at 30 °C, and the absorbance was then measured at 517 nm with a FLUostar OPTIMA spectrophotometer (BMG Labtech, Germany).

For cytotoxicity assay, 100 µl of cell suspension was seeded into a 96-well plate (2 × 10⁴ cells/well) and allowed to adhere overnight. On the following day, the cells were incubated with BAs then 100 µl supernatant from each of the wells was carefully transferred into a new 96-well plate containing 100 µl reaction mixture. We then measured lactate dehydrogenase (LDH) activity at 490 nm using a FLUOstar OPTIMA Spectrophotometer (BMG Labtech, Ortenberg, Germany). For background controls, we measured 200 µl assay medium, without cells. For low controls, we used 100 µl cell suspension and 100 µl assay medium. In the case of high controls, the mixture of 100 µl cell suspension and 100 µl Triton-X 100 (0.1%) solution was measured. The LDH release induced by Triton-X 100 was assigned to 100%. The average absorbance values of each of the triplicates were calculated and the average value of the background control (LDH activity contained in the assay medium) was subtracted from each of the samples to reduce background noises. We then calculated the percentage of cytotoxicity using the following formula: Cytotoxicity (%) = (exp. value–low control/high control-low control)*100. Low control determines the LDH activity released from the untreated normal cells (spontaneous LDH release), whereas high control determines the maximum releasable LDH activity in the cells (maximum LDH release).

HDFs were cultured in 96-well plates in Dulbecco’s Modified Eagle Medium (DMEM) supplemented in 10% fetal bovine serum (FBS) in a 5% CO₂ humidified environment until 60–75% confluent. Serial dilutions of galunisertib (0 µM, 0.01 µM, 0.1 µM, 1 µM, 2.5 µM, 10 µM, and 100 µM), together with a 1% DMSO and 5% DMSO control, were added to the FPDF and HDF cell cultures. FPDFs were incubated with galunisertib for 0, 24, 72, and 168 h without medium exchange. After incubation, 10 µL of MTT reagent (ATCC 30-1010K kit) was added to each well with a 2 h incubation time. A proprietary detergent reagent (100 µL) was added, and cells were allowed to incubate for 2 h at room temperature. The absorbance at 570 nm was then measured using a BMG LabTech Fluostar Optima spectrophotometer.