Cell culture dishes

Small intestinal tissue from n = 14 guinea pigs was used to obtain primary culture of enteric neurons as described elsewhere (Kugler et al., 2015, 2018).
Briefly, after mechanical separation of longitudinal muscle-myenteric plexus preparation and following enzymatic digestion, cell culture dishes (Ibidi) were seeded with 200 µL of myenteric ganglia suspension. This was incubated in medium M199 supplemented with 10% fetal bovine serum (FBS) (Gibco), 50–100 ng mL⁻¹ mouse nerve growth factor 7S (Alomone labs, Jerusalem, Israel), 5 mg mL⁻¹ Glucose, 100 U mL⁻¹ Penicillin, 100 mg mL⁻¹ Streptomycin (Gibco) and 2 mM arabinose-C-furanoside (Sigma–Aldrich, St. Louis, MO, USA). The neurons were cultured in vitro under standard culture conditions (5% CO₂; 37 °C; humidity 95%). Medium with additives was changed every second day. The cultures were grown for at least two weeks to obtain interconnected neuronal clusters.

1 × 10⁵ HT1080 and RPE-1 cells per well were plated on ibidi-cell culture dishes (Ibidi, United States) and treated with PLE extract at LC₅₀ concentration for 24 h. As a positive control, Etoposide was used at concentration 1 μM. Cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature, then permeabilized with 0.2% Triton X-100 in PBS for 10 min. Cells were blocked with 5% milk in TBS for 2 h at room temperature and treated with rabbit monoclonal anti-phospho γ-H2AX (Ser139) antibody (Abcam, United States) in 1:500 dilution with 5% milk/TBS at 4°C overnight. Goat Anti-Rabbit Alexa Fluor^® 568 conjugated secondary antibodies were used in 1:500 dilution with 5% milk/TBS at room temperature for 2 h. Cells were stained with DAPI (diluted 1:15,000 in PBS) for 10 min at room temperature. Quantification of the γ-H2AX foci in nuclear was performed using the FV1200 confocal laser scanning microscopy system equipped with the objective lens (UPLSAPO ×100) (Olympus, Japan). A 405 nm LD Laser with Integrated Transmitted Light Photomultiplier Detector and 488 nm Argon Laser with High-Sensitivity Detector (GaAsP) were used. To avoid cross detection, the images were acquired sequentially at 488 nm (Argon) and 405 nm (LD). All taken Z-sections were condensed into one single plane in order to visualize all detectable foci.

Cytokinesis-block micronucleus formation assay was performed as described earlier (Fenech 2007) with minor changes: 1 × 10⁵ HT1080 or RPE-1 cells per well were plated on ibidi-cell culture dishes (ibidi, United States) and treated with PLE extract at LC₅₀ concentration for 24 h. Cells were fixed with 3.7% formaldehyde in PBS for 10 min at room temperature. Scoring Procedure Cells were stained using 15 μl of a DNA-specific stain, namely 40,6-diamino-2-phenylindole dihydrochloride (DAPI). About 2,000 cells were scored using the FV1200 confocal laser scanning microscopy system equipped with the objective lens (UPLSAPO ×100) (Olympus, Japan) and 405 nm LD Laser with Integrated Transmitted Light Photomultiplier Detector. The criteria for evaluation of micronucleus formation assay were: 1) cell integrity (intact nucleus and cytoplasm); 2) similar staining of nucleus and MNi; 3) nucleus and MNi are in the same plate. The frequency of cells with micronuclei (MNi) and nucleoplasmic bridges (NPBs) was determined for each analyzed subject.