Ethidium homodimer 1 ethd 1

Bacterial suspensions (adjusted to optical density at 600 nm = 0.2) of MDR A. baumannii Ab186 and MDR E. coli EcMCR+ strains were placed on a 96-well plate, incubated with 2 and 16 mg/L of tamoxifen metabolites mixture, respectively, and mixed in a solution of phosphate buffered saline containing Ethidium Homodimer-1 (EthD-1) (1:500) (Invitrogen, Carlsbad, CA, USA). After 10 min of incubation, fluorescence was monitored during 160 min using a Typhoon FLA 9000 laser scanner (GE Healthcare Life Sciences, Marlborough, MA, USA) and quantified with ImageQuant TL software (GE Healthcare Life Sciences, USA). Bacterial counts were obtained at the beginning and end of the experiment to ensure that the metabolite mixture did not present bactericidal activity against A. baumannii and E. coli strains.

The live/dead^® viability kit for mamalian cells (Invitrogen, Paisley, UK) was used to stain the live and dead cells of the microtissues formed in the calcium alginate microcapsules. The live/dead^® cell viability kit can differentiate live cells from the dead cells by double staining of both the HaCaT and ORL-48 cells in the microtissues with green-fluorescent Calcein-Acetoxymethyl (AM, Invitrogen, Paisley, UK), which indicates intracellular esterase activity, and red-fluorescent Ethidium homodimer-1 (EthD-1) (Invitrogen, Paisley, UK), which indicates the loss of plasma membrane integrity. After 16 days of culture, the HaCaT and ORL-48 microtissues formed in the calcium alginate microcapsules were incubated in 2 μM of Calcein-AM and 4 μM of EthD-1 stain solutions for 20 min in the dark. Subsequently, the stain solutions were removed and the microtissues were washed three times in HBSS solution. The stained microtissues were captured using a BX53 fluorescence microscope (Olympus, Tokyo, Japan) mounted with a DP73 digital camera.

For drug treatment, lung cancer cells (NCI-H460, A549, and SK-MES-1) and stromal cells (WI38 and HUVEC) were seeded at a density of 6 × 10³ cells/well in 96-well round-bottomed ultra-low attachment microplates. After 1 day, 10 or 20 μM of Gefitinib and Cisplatin (all from Sigma-Aldrich, St. Louis, MO, USA) were added to the spheroids for 2 days, and then spheroid cell death was detected using the cell-impermeant viability indicator ethidium homodimer-1 (EthD-1; Invitrogen Life Technologies, Grand Island, NY, USA). EthD-1 is a high-affinity nucleic acid stain that fluoresces weakly until bound to DNA, whereupon it emits red fluorescence (excitation/emission maxima ~ 528/617 nm). Spheroids were incubated in 4 μM EthD-1 in complete medium for 30 min in a 37 °C incubator, and images were obtained and the intensity of EthD-1 fluorescence measured using the Operetta® High Content Screening System (Perkin Elmer).

For the fabrication of array chips for DCST, poly(dimethylsiloxane) (PDMS) monomers and curing agents were purchased from Dow Corning (Midland, MI, USA). Poly(methyl methacrylate) (PMMA, 3 mm thickness) plates were purchased from YM Tech (Daejeon, Korea). Phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S) were purchased from Corning (Corning, NY, USA). Rat tail collagen type I was purchased from Corning. Calcein-AM and ethidium homodimer-1 (EthD-1) and Hoechst 33342 solution were purchased from Invitrogen (Carlsbad, CA, USA). CellTracker^TM Green CMFDA and CellTracker^TM Red CMTPX dyes for cell tracking were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Doxorubicin hydrochloride for drug treatment was purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals and reagents were purchased from Sigma-Aldrich unless otherwise stated.