Uplsapo 100

The SPEED microscope included an Olympus IX81 equipped with a 1.4-NA 100×oil-immersion apochromatic objective (UPLSAPO 100×, Olympus), a 35 mW 633 nm He-Ne laser (Melles Griot), 50 mW solid state 488-nm and 561-nm lasers (Coherent), an on-chip multiplication gain charge-coupled-device camera (Cascade 128 + , Roper Scientific) and the Slidebook software package (Intelligent Imaging Innovations) for data acquisition and processing. For individual channel imaging, GFP (or mCitrine), mCherry, and Alexa Fluor 647 were excited by 488 nm, 561 nm, and 633 nm lasers, respectively. The fluorescence emissions were collected by the same objective, filtered by a dichroic filter (Di01- R405/488/561/635-25 × 36, Semrock) and an emission filter (NF01- 405/488/561/635-25 × 5.0, Semrock) and imaged with the above CCD camera operating at 500 Hz.

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.