Sybr green 1

The comet assay was performed using a Trevigen Comet Assay Kit (Trevigen Inc., Gaithersburg, MD, USA) to detect DNA strand breaks at the single-cell level as described previously [18 (link)]. Briefly, cells were exposed to millimeter-wavelength radiation for 24 h, collected by trypsinization and centrifuged immediately after exposure, then mixed with low melting point agarose to prepare a cell suspension in 0.1% agarose/phosphate buffered saline (PBS). After gelation of the agarose, the cells were lysed, then the alkaline unwinding was performed (1 h at 4 °C, pH > 13). The resulting DNA samples were electrophoresed at 1 V/cm for 30 min in a 0.3 M NaOH (Nacalai Tesque, Kyoto, Japan) and 1 mM ethylenediamine-N,N,N’,N’-tetraacetic acid (Sigma-Aldrich) solution. After the DNA was stained with SYBR Green I, immunofluorescence images were captured using a fluorescence microscope (Olympus, Tokyo, Japan). DNA strand breaks were analyzed using Comet software (Perceptive Instruments, Suffolk, UK). At least 100 comets from each gel were analyzed, and at least five independent experiments were performed. Tail length indicates the pixel length of the comet tail, tail percent indicates the percentage of tail content relative to comet content, and tail moment was calculated as follows:

For the detection of cells in the S phase, seedlings 3 d after germination (DAG) were placed in liquid medium containing half-strength-MGRL and 10 μM 5-ethynyl-2′-deoxyuridine (EdU) in Click-iT component A (Invitrogen) for 30min at 22 °C under continuous light. EdU incorporation was stopped by fixation with 4% PFA/PBS for 30min under vacuum. After three washes with PBS, the seedlings were incubated with 0.5% Triton X-100/PBS for 20min. After three washes with PBS, EdU was labelled with Alexa Fluor 594 azide following the manufacturer’s instructions. Nuclei were stained with SYBR Green I (Lonza) diluted 5 000-fold with 0.5% Triton X-100/PBS for 10min. The seedlings were then mounted with 1/2× mounting medium as described in Hayashi et al. (2013) . Fluorescence emitted from Alexa Fluor 594 and SYBR Green I was observed using a fluorescent microscope (IX-81, Olympus) equipped with a confocal scanning unit (CSUX-1, Yokogawa) and a sCMOS camera (Neo 5.5 sCMOS ANDOR Technology). The excitation and emission wavelengths were 561nm and 604–644nm for EdU and 488nm and 503–537nm for SYBR Green I, respectively. Images were analysed using ImageJ software. M phase cells were distinguished from other cells, based on SYBR Green I staining, with obvious features of prophase, metaphase, anaphase, and telophase.