Plan fluor 40x objective lens

Live imaging of cells stained with H₂DCFDA and JC-1 was performed using a confocal microscope system (A1R, Nikon, Tokyo, Japan) equipped with a PlanFluor 40X objective lens (N.A. 0.6, W.D. 3.7–2.7 mm, Nikon) and excitation lasers (488 and 561 nm, Melles Griot). To measure the ROS level, a stage incubator (37°C and 5% CO₂) was utilized. For immunocytochemical study and TUNEL assays, images were obtained with an AxioImager M2 epifluorescence microscope equipped with 20X and 40X objective lens, an AxioCam HR camera, an excitation filter BP379-401; dichromatic filter FT 420; emission 435–485; under the control of AxioVision rel. 4.8 software (Carl Zeiss, Jena, Germany). Images were saved in TIFF format and analyzed by ImageJ software (Wayne Rasband, National Institutes of Health). For quantification, images were taken of at least five randomly selected fields for each sample, and fluorescence intensity in at least 24 cells was quantified per sample in three independent experiments. Areas without cells in each sample were considered as background and their intensity was subtracted from fluorescence intensity.

To visualize intracellular sgRNA, HSC-3 cells (1×10⁵ cells/well) were seeded into collagen-coated glass-bottomed dishes (Matsunami glass Inc., Osaka, Japan). After 24 h, the cells were treated with 200 nM naked Alexa568-3′-labeled sgRNA, and then cultured for a further 24 h. The cells were then rinsed twice with 1× phosphate-buffered saline (PBS), and then an inverted microscope (Nikon, Ti-E, Tokyo, Japan) equipped with a Plan Fluor 40x objective lens (NA 0.75, Nikon) or a Plan Apo VC 100x objective lens (NA 1.40, Nikon) and micro scanning stage (BI XY stage, Chuo Precision Industrial Co. Ltd., Tokyo, Japan) was used to observe fluorescence images in living cells maintained at 37°C with a continuous supply of 95% air and 5% CO₂ using a stage-top incubator (INUBG2TF-WSKM, Tokai Hit, Fujinomiya, Japan). The nuclei or mitochondria were visualized with Hoechst 33342 (H21492, Molecular Probes, Invitrogen, Eugene, OR) or MitoTracker Green FM (Molecular Probes), respectively. The fluorescent cells were counted randomly at least 15 fields under the fluorescence microscope with a 40× objective lens and calculated as a percentage of the total number of fluorescent cells.

The cells were labeled with 2 µM CellEvent Caspase-3/7 green detection reagent (Life Technologies) which is a nucleic acid-binding dye that harbors the caspase-3/7 cleavage sequence, DEVD, and fluoresces after being cleaved and bound to DNA. After incubation for 30 min at 37°C in a humidified atmosphere of 5% CO₂, fluorescence images were observed by an inverted microscope (Nikon, Ti-E) equipped with a Plan Fluor 40x objective lens (NA 0.75, Nikon) maintained at 37°C with a continuous supply of 95% air and 5% CO₂ using a stage-top incubator (INUBG2TF-WSKM, Tokai Hit). Images were captured using a cooled CCD camera (ORCA-R2, Hamamatsu Photonics).
Cellular enzymatic activities of caspases 3/7 were determined by a caspase colorimetric assay (Caspase-Glo 3/7 Assay Systems, Promega, Madison, MI) according to the manufacturer's instructions. Briefly, for each reaction, cells were lysed and incubated with a luminogenic substrate containing the DEVD sequence, which is cleaved by activated caspase 3/7. After incubation at room temperature for 3 h, luminescence was quantified with a luminometer (Glomax 20/20, Promega).