Ethd 1

Promastigotes (1 × 10⁶, 200 μL) were incubated with NET-rich supernatants (0.5 µg/mL) for 2 h at 35 °C, 5% CO₂, and cell viability was assessed with 4 μM ethidium homodimer-1 (EthD-1, Molecular Probes, Eugene, OR, USA) staining solution for 30 min. Promastigotes killed by 4% formaldehyde served as the positive control. The data were collected with a FACS Calibur flow cytometer and analyzed with FlowJo software.

The RVECs (1 × 10⁴ cells/well) were grown in 96-well plates and exposed to serially-diluted moxifloxacin (Vigamox^®, Alcon, Fort Worth, TX), cefuroxime (GlaxoSmithKline, Brentford, UK), or vancomycin (Meiji, Tokyo, JPN) in serum-depleted medium for 24 hours. The cell membrane damage caused by these antibiotics allowed ethidium homodimer-1 (EthD-1; Molecular Probes, Eugene, OR) to enter the cells and bind to the DNA. To determine the degree of damage, the amount of bound EthD-1 was measured with a fluorescent microplate reader (Tecan, Männedorf, Switzerland) with excitation by 495 nm light and emission at 635 nm. Saponin (0.1%, Sigma, St. Louis, MO)-treated RVECs were used as positive control of dead cells.
We measured the cell viability by the esterase activity because viable cells have intrinsic esterase activity. To measure the esterase activity, antibiotics-exposed RVECs were exposed to non-fluorescent calcein AM (Molecular Probes, Eugene, OR), which is converted to fluorescent calcein by the esterase activity of living cells. The intensity of the fluorescence was measured with a microplate reader with excitation by 495 nm and emission at 530 nm.
The cell viability and membrane damage were calculated by the following formula, % alteration = (antibiotics-treated cell emission – non-treated cell emission)/(Positive control cell emission – non-treated cell emission) × 100.

Acrosome integrity was evaluated using EthD-1 (Fertilight^®, Sperm Viability Kit, Molecular Probes Europe, Leiden, Netherlands). Briefly, 10 μl of aliquot of the sperm sample was thoroughly mixed with 10 μl of 14-μM EthD-1 (Molecular Probes Inc., OR, USA) at 37°C for 15 min. Five microliters of the mixture was placed on a glass slide and dropped into 95% ethyl alcohol for 30 s and then added to 15 μl FITC-PNA solution [FITC-PNA in PBS (1:10, v/v)] at 4°C for 30 min and removed by PBS. Acrosome integrity was assessed using an epifluorescent microscope (CX-31; Olympus, Tokyo, Japan) at × 1,000 magnification. Two hundred sperm were evaluated in five different areas.

Equal numbers of JIMT-1 and DU145 cells were dispersed into 96-well plates. After 24 h the cells were incubated with varying concentration of compounds 3 or 4 (4–6 µM) for 5–6 h. Subsequently, 2 µM of the cell membrane impermeant dye, ethidium homodimer-1 (EthD-1 (Molecular Probes, Eugene, OR, USA) that emits red fluorescence upon binding to DNA was added and the cells analysed by fluorescence microscopy. EthD-1 staining was compared to negative controls with no treatment and positive control treated with 0.01% Triton X-100 for 10 min.

Sperm vitality was evaluated using SYBR-14/ethidium homodimer-1 (EthD-1) (Fertilight^®, Sperm Viability Kit, Molecular Probes Europe, Leiden, Netherlands). Briefly, 10 μl of aliquot of the sperm sample was thoroughly mixed with 1 μl of 14-μM EthD-1 (Molecular Probes Inc., OR, USA) in 1 ml PBS and 2.7 μl of 0.38-μM SYBR-14 (Dead/Alive Kit; Molecular Probes Inc.) in 1 ml dimethyl sulfoxide (DMSO) at 37°C for 15 min. The sperm was placed on a glass slide and covered with a coverslip. Sperm membrane integrity was assessed using an epifluorescent microscope (CX-31; Olympus, Tokyo, Japan) at × 1,000 magnification. Two hundred sperm were evaluated in five different areas and classified into two categories: live and dead sperm which were stained only green from SYBR-14 (live) and stained both green and red or stained only red from EthD-1 (dead). The percentages of intact sperm membrane were calculated.

The viability of ECs and SCs in both mono- and cocultures was studied at d 1, d 7, and d 14 time points with qualitative live/dead viability staining as described previously [13 (link), 16 (link)]. The samples were incubated at 0.5 mM CalceinAM (green fluorescence; Molecular Probes) and 0.25 mM EthD-1 (green fluorescence; Molecular Probes) in DBPS (Sigma-Aldrich) for 45 min at RT and imaged with a fluorescence microscope (Olympus IX51S8F-2; camera DP71, Tokyo, Japan). The viable cells were stained green, and the dead cells were stained red. The background fluorescence caused by the scaffold without cells was used as a negative control.
The CyQUANT™ cell proliferation assay kit (Invitrogen) measuring the total DNA amount was used to evaluate the proliferation of ECs and SCs in monocultures at d 1, d 7, and d 14 as previously described[16 (link)]. Briefly, the cells were lysed with 0.1% Triton-X-100 buffer (Sigma-Aldrich) and stored at − 70 °C until analysis. After the freeze–thaw cycle, the working solution containing CyQUANT™ GR dye and cell lysis buffer was added, and the fluorescence at 480/520 nm was measured with a Victor 1420 Multilabel Counter microplate reader (Wallac, Turku, Finland).