Chamber slide

To monitor epidermal cell morphogenesis, time-lapse imaging of the cotyledon surface was performed with A. thaliana seedlings as described previously [20 ]. Sterilized seeds expressing the plasma membrane marker GFP-PIP2a [21 (link)] were immersed in distilled water at 4°C for 2 days, and the seed coats were then carefully removed under a stereo microscope (SZX12, Olympus, Tokyo, Japan). The naked cotyledons were mounted on a chamber slide (Iwaki Co., Ltd, Tokyo, Japan) and covered with 1/2-strength Murashige–Skoog medium agar gel (2.3 g L⁻¹ Murashige and Skoog Plant Salt Mixture, pH 5.8 from Wako Pure Chemical Industries, Osaka, Japan). The chamber slides were placed in growth chambers at 23.5°C, with a 12-h light/12-h dark cycle, using 100 μmol m⁻² s⁻¹ white light. For acquiring images, the chamber slide was placed onto the inverted platform of a fluorescence microscope (IX70, Olympus) equipped with a UPlanFl 20×/0.50 objective lens and spinning disc confocal unit (CSU10, Yokogawa Electric Co., Ltd, Tokyo, Japan), together with a cooled CCD camera head system (CoolSNAP HQ; Photometrics, Huntington Beach, Canada).

Kidney cells were harvested with 0.1% trypsin and 0.02% EDTA in a balanced salt solution (Nacalai Tesque) and plated onto a chamber slide (Iwaki, Tokyo, Japan) using their corresponding media without antibiotics. The slides were incubated for 48 h until a monolayer was formed and washed twice with media to remove non-adherent cells. The cells were incubated for a further 2 h at 37°C and 5% CO₂. Approximately 500 μL of stationary phase Leptospira cells were harvested by centrifugation at 1,000 × g for 10 min at room temperature, washed twice in PBS, then resuspended in the corresponding kidney cell culture media without antibiotics at 37°C to a concentration of 10⁷ cells/mL. These suspensions (1 mL) were then added into the corresponding chamber slides containing the kidney cell layer, and the chamber slides were incubated at 37°C for 1 h.

In vivo: after the confirmation of tumor induction in all groups except the cancer-free mice drinking hydrogen water, mice were treated every 8 h with MQ water (Milli-Q Integral per produce), HHW or NHW by oral administration (250 µl), and 5-FU dissolved in a 5% glucose solution was administered (100 mg/kg) by intraperitoneal injection weekly (Perboni et al., 2008 (link)). At ten days after tumor induction, the mice were sacrificed, and the tumors were excised and scaled. Tumor size is measured with caliper and calculated using the following equation: Length × width² × 0.5, as previously described (Lee et al., 2012 (link)).
In vitro A solution of 50 µl MQ, HHW (0.8 mM) or NHW (0.125 mM) was added to each slide chamber (IWAKI, Japan), and the results were compared with slides chambers administered only 5-FU at a final dose of 50 µM.

Cells (5 × 10⁴ cells) seeded on slide chamber (Iwaki, Tokyo, Japan) were incubated for 6 h, and then irradiated with X-rays. After four days of culture, the cells were washed with PBS(-) and fixed with 4% paraformaldehyde/PBS(-) for 5 min at room temperature. The cells were washed three times with PBS(-) and treated with 1% TritonX-100/PBS(-) for 20 min at room temperature. After three washes with PBS(-), the slides were sealed with Antifade Mounting Medium with DAPI (VECTASHIELD, LSBio). The slides were photographed using a fluorescence microscope (IX71, Olympus, Tokyo, Japan), and the nuclear morphology was evaluated. Cells with abnormal mitotic nuclear features such as micronuclei, multilobular nuclei, and fragmented nuclei were scored as cells in mitotic catastrophe [22 (link)]. More than 100 cells were counted for each condition.