Fixdenat

Cells are cultured in the presence of the respective test substances in a 96-well MP at 37 °C for a certain period of time (1–5 days, depending on the individual assay system); subsequently, BrdU is added to the cells and the cells are reincubated (usually 2–24 h). During this labeling period, the pyrimidine analogue BrdU is incorporated in place of thymidine into the DNA of proliferating cells; After removing the culture medium the cells are fixed and the DNA is denatured in one step by adding FixDenat (Roche, CH, Basel, Switzerland) (the denaturation of the DNA is necessary to improve the accessibility of the incorporated BrdU for detection by the antibody); The anti-BrdU-POD binds to the BrdU incorporated in newly synthesized, cellular DNA; the immune complexes are detected by the subsequent substrate reaction; and the reaction product is quantified by measuring the absorbance at the respective wavelength using a scanning multiwell spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The developed color and thereby the absorbance values directly correlate to the amount of DNA synthesis and thereby to the number of proliferating cells in the respective microcultures.

5-Bromo-2-deoxyuridine (BrdU) incorporation into DNA was studied with the BrdU “Cell Proliferation Elisa BrdU colorimetric” (Roche, Basel, Switzerland). 300–500 cells were seeded by Cell Sorter (BD FACSAria) into 96-well plates on a Matrigel layer, preparing 3–6 replicates per experimental condition. Briefly, cells were exposed to BrdU (1:100) for 16 h before the end of exposure to hypoxia or DETA/NO. Subsequently, cells were fixed and exposed to the anti-BrdU antibody (1:100) for 2 h at RT. To allow the antibody to bind to the BrdU incorporated into the DNA, cells must be pre-fixed, permeabilized and DNA-denatured through Kit fixing solution (Fixdenat, Roche) for 30 min at RT. To remove the excess antibody, 3 washes with 200 μL of wash solution included in the kit were performed. Cells were then incubated with the substrate solution for 5–30 min at RT to finally stop the reaction with H₂SO₄. Finally, absorbance at 450 nm was measured which is proportional to the degree of cell proliferation.

BrdU assay was carried out using a cell proliferation ELISA BrdU kit (Roche Diagnostics) according to the manufacturer's recommendations. In brief, following the treatment period with the assigned concentrations of the essential oils, 100 µl BrdU solution (100 µM) were added to each well in 1 ml medium, and the plates were then incubated at 37°C for 4 h. Subsequently, the cells were fixed using 1 ml FixDenat (Roche Diagnostics) in each well for 30 min and incubated with the kit-supplied anti-BrdU antibody (1:100; Roche Diagnostics) for 90 min at room temperature. After washing, the cells were incubated with 500 µl substrate for 20 min at room temperature, and 125 µl H₂SO₄ (1 M) were added. The plates were analyzed at 450 nm using a spectrometer (Agilent Technologies, Inc.).

Three different siRNAs were designed against each of the 286 candidate lncRNAs to avoid off-target effects, and transfected into PC3 and MCF10A cells using three 96-well plates with three technical replicates. Two to three days after transfection, cells were incubated with 10 µM BrdU for 15 min to 1 hr, and fixed with FixDenat (Roche, Indianapolis, IN) for 30 min. Cells were blocked with 3% BSA in PBS for 1 hr, then incubated with HRP-coupled anti-BrdU antibody (Roche) diluted in 3% BSA in PBS for 1 hr. After washing three times with PBS containing 0.1% TX-100, cells were incubated with TMB substrate (Pierce, Rockford, IL) for 5–10 min and analyzed by the absorbance at 450 nm following the addition of 1 M H₂SO₄ to stop the reaction. For data analysis, each assay plate contained four wells of negative control (luciferase; GL2) and two wells of positive control (ORC2) siRNAs for normalization. To normalize values of BrdU incorporation, we calculated the inhibition index (%) using the following equation: Inhibition index of gene X (%)  =  (GL2av−X)/(GL2av−ORC2av) ×100, where X, GL2av, and ORC2av represent BrdU incorporation (absorbance at 450 nm) of gene X and average of GL2 and ORC2, respectively. Genes with a SD of inhibition indices (from 3 technical replicates) greater than a cutoff value were eliminated to select technically reproducible data.

Cell proliferation was measured with the commercial Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, cells were cultured in 96-well plates at a density of 5 × 10³ cells in normal culture medium (100 μL) for 24 h, after which the culture medium was changed and the cells exposed to 0.2, 1, 5, 10, and 25 mg/mL of bilberry. PBS (25 µL/100 µL) was used as a control. After 24- and 72-h incubations, the cells were labelled using 10 μM BrdU per well and re-incubated for 2 h at 37 °C in a humidified atmosphere. Culture medium was removed, cells were fixed, and DNA was denatured in one step by adding FixDenat (Roche Diagnostics, Basel, Switzerland). Next, the cells were incubated with anti-BrdU-POD for 90 min at room temperature. After removal of the antibody conjugate, cells were washed twice, and substrate solution was added. The reaction product was quantified by measuring absorbance using a scanning multi-well spectrophotometer (Thermo Scientific Multiskan EX, Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm with a reference wavelength of 690 nm. There were two (HSC-3 cells) or three (HMK and IHGK cells) independent experiments with six replicates in each assay.