Pathway 855 confocal microscope

Cells were plated in 96-well clear bottom black plates at 1 × 10⁴ cells per well and allowed to adhere for 24 h. Cells were treated with the respective concentration of flavonoids and incubated for 48 h. Medium was removed and cells were fixed at room temperature for 15 min with 4% paraformaldehyde, rinsed three times with PBS, and permeabilised at room temperature for 5 min with 0.1% Triton X-100. Then, non-specific binding was blocked with 10% heat-inactivated goat serum in PBS for 1 h at room temperature. Cells were incubated with the following antibodies at 4°C, overnight: anti-p53 antibody (sc-126, 1:200) and anti-Nrf2 antibody (sc-365949, 1:200) obtained from Santa Cruz Biotechnology, and anti-cleaved-caspase-3 antibody (#9664, 1:200) and anti-cleaved-caspase-7 antibody (#8438, 1:200) purchased from Cell Signaling Technology. After three washes with PBS, cells were incubated with Alexa Fluor 594 conjugated antibody (A11032 or A11037; Invitrogen) for 1 h in the dark. Cell nuclei were stained with Hoechst 33342 (Invitrogen) and F-actin was stained with Phalloidin-Atto 488 (Sigma-Aldrich). Fluorescent signals were examined and captured using a BD Pathway 855 confocal microscope (Becton Dickinson). The mean fluorescence intensity was calculated using ImageJ software (National Institutes of Health).

Firstly, to examine fluorescent signals, cells were placed in the bottom of 96-well black plates at 1 × 10⁴ cells per well and permitted to adhere for 24 h. Subsequently, the cells were treated with compounds 1, 6, 15–17, and 37 at concentrations of 25 μM, 50 μM, or 25 μM cPt for 48 h. After incubating and removing the medium, cells were fixed in 4% paraformaldehyde for 15 min. Later, cells were rinsed three times with PBS and permeabilized with 0.1% Triton X-100 for 5 min at room temperature. The 10% heat-inactivated goat serum in the PBS was used to block non-specific binding. Subsequently, cells were treated with the following antibodies overnight: anti-cleaved caspase 3 and anti-cleaved caspase-7. Next, cells were incubated with the Alexa Fluor 594 conjugated antibody for 1 h in a dark place. Cell nuclei were stained with Hoechst 33342 and F-actin was stained with Phalloidin-Atto 488. Fluorescent signals were captured using a BD Pathway 855 confocal microscope (Becton Dickinson, Franklin Lakes, NJ, USA). The mean fluorescence intensity was calculated using ImageJ software (National Institutes of Health, Bethesda, Rockville, MD, USA).

Cells were seeded in BD Falcon™ 96-well black, clear-bottom tissue culture plates at 10,000 cells per well. These plates are optimized for imaging applications. Analyses were performed in duplicate in three independent experiments. After incubation, cells were rinsed with PBS and fixed with a 3.7% formaldehyde solution at room temperature for 10 min. Cells were then washed three times with PBS and permeabilized with 0.1% Triton X-100 at room temperature for 5 min. Next, the cells were washed twice with PBS, and nonspecific binding was blocked by incubation in 3% FBS at room temperature for 30 min. The cells were rinsed and incubated with either anti-Nrf2 rabbit polyclonal antibodies (Sigma-Aldrich, St. Louis, MO, USA; 1 : 1000) or anti-NFκB (p52) mouse polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Cells were then washed three times with PBS and incubated with FITC-conjugated anti-rabbit secondary antibodies (BD Pharmingen, San Diego, CA) for 60 min in the dark. After washing, nuclei were stained with Hoechst 33342 (2 μg/mL) and analyzed using a BD Pathway 855 confocal microscope with a 40x (0.75 NA) objective. The cytoplasmic and nuclear fluorescence intensities of stained cells were analyzed, and images of FITC-labeled cells were acquired using a 488/10 excitation laser and a 515LP emission laser.