Microplate spectrofluorometer biotek synergy

ROS
generation was assessed by the spectrofluorometric method using
the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA)
or dihydrorhodamine 123 (DHR-123). The method is based on the ROS-dependent
oxidation of the compounds to fluorescent dichlorofluorescein (DCF)
or rhodamine-123, respectively.^{61 (link)} PC3,
SW480, SW620, and HaCaT were seeded on to 96-well plates (5 ×
10⁴ cells per well) and allowed to adhere for 24 h. Then,
cells were rinsed with PBS and incubated with DCFH-DA (5 μM)
or DHR-123 (1 μM) for 30 min at 37 °C in the dark. Thereafter,
cells were rinsed with PBS and treated for 1, 4, 12, 24, and 72 h
at 37 °C with red phenol free culture medium containing compound 12 or 13 at their IC₅₀ concentrations
to observe the level of ROS. A sample with H₂O₂ (1.5 mM) was a positive control, and a sample without any reagent
was a negative control. Maximum excitation and emission spectra for
DCF were 492 and 527 nm, and those for rhodamine-123 were 500 and
536 nm, respectively. The generation of H₂O₂ was measured by Microplate Spectrofluorometer BioTek Synergy (BioTek
Instruments, USA) and expressed as fluorescence intensity (FI). Values
from three experiments performed in triplicate were analyzed.

ROS generation was performed using the spectrofluorometric method using dihydrorhodamine 123 (DHR 123) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). The following analysis is based on ROS-dependent oxidation of the compounds to fluorescent rhodamine 123 and dichlorofluorescein (DCF), respectively. K562 cells were seeded to a 96-well plate (1 × 10⁴ cells) and the selected benzofuran compounds (6 and 8) were added according to their respective IC₅₀. The incubation times were 6 and 12 h, respectively. Then, the medium was removed, and cells were rinsed with PBS and incubated with DHR 123 (5M) and DCFH-DA (5M) for 60 min at 37 °C in the dark. In order to obtain a positive and negative control, a sample with H₂O₂ (0.25 mM) and a sample without any reagent were prepared, respectively. The maximum excitation and emission spectra for rhodamine 123 were 500 nm and 536 nm, respectively, and for DCF were 492 nm and 527 nm, respectively. The generation of H₂O₂ was measured by a Microplate Spectrofluorometer BioTek Synergy™ (BioTek Instruments, USA) and expressed as DCF fluorescence intensity (FI) and rhodamine fluorescence intensity (FI). The resulting data were expressed as mean ± SD from three independent experiments performed in triplicate.

The induction of cell apoptosis was also analyzed by luminescent assay with the Caspases-Glo3/7 assay system (Promega). K562 and HaCaT cells (1 × 10⁴/well) were seeded onto a 96-well plate. After 24 h, cells were exposed to compounds 6 and 8 and their respective IC₅₀ concentrations. Cells were incubated for 4, 6, and 12 h. After incubation, cells were treated with caspase 3/7 reagent (according to manufacturer’s instruction) and incubated for a further 1.5 h at room temperature. Fluorescents of the wells (performed three times for each well) were measured at the excitation wavelength of 485nm and emission wavelength of 520nm using a Microplate Spectrofluorometer BioTek Synergy™ (BioTek Instruments, Santa Clara, CA, USA).