Arvo multilabel counter

The cytostatic activity of representative aptamers against each cell line was examined through WST-8 assays, as follows. Aliquots (0.1 mL) of each cell solution (1–2.5 × 10⁴ cells per milliliter for cancer cell lines, except for KG-1, and 4 × 10⁴ cells per milliliter for HUVEC and MCF10A cells) were plated into each well of a 96-well microplate, 1 day before the addition of DNA aptamers. In the case of KG-1 cells, 90 μL cell solution was plated into each well, 4 hr before the DNA addition. Each DNA aptamer was folded in D-PBS (−), similar to the DNA library used for cell-ExSELEX, at a 0.1mM concentration. The folded DNA aptamer, as well as FUdR (Nacalai Tesque), was used as a positive drug control and was diluted with D-PBS (−) and the medium used for each cell line, and added to the cells at final concentrations of 1, 2.5, 5, or 10 μM for DNA and 10 or 100 nM for FUdR in 10% D-PBS (−)/media (0.1 mL per well). As another positive control, PTX (Wako Pure Chemical Industries) in DMSO was also used for the analysis and added at the final concentration of 10 nM in 0.1% DMSO/media (0.1 mL per well). After incubation for 96 hr, 10 μL Cell Count Reagent SF (Nacalai Tesque) was added to each well, and the absorbance of the formazan product obtained by WST-8 reduction was measured at 450 nm with an ARVO multilabel counter (PerkinElmer).

TKE2 were seeded in DKSFM culture medium at a density of 1.0 × 10⁴ cells/well in 96-well plate, at 37°C, in 5% CO₂ and cultured overnight. Intracellular reactive oxygen species (ROS) levels were detected by using 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-DCFH₂DA; Life Technologies). CM-DCFH₂DA and quercetin were each diluted in dimethyl sulfoxide (DMSO: Wako pure chemical, Osaka, Japan) to obtain 1 mM stock solutions, which were serially diluted to make working solutions prior to application to TKE2 cells. Treatments with CM-DCFH₂DA were for 1 h, followed by washes with PBS to remove extracellular CM-DCFH₂DA. The cells were then stimulated by H₂O₂ for 1 h, before incubation with culture medium containing quercetin for 1 h. After incubation, the fluorescence (excitation 485 nm, emission 538 nm) in each well was measured using an ARVO multilabel counter (PerkinElmer; Boston, MA, USA).