Cells were plated and cultured in 24-well plates. After drug treatment for 48 h and labeled with Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ) as followed manufacturer’s instructions. In brief, 100 μL of binding buffer containing annexin-V/FITC and propidium iodide (PI) were used for covering cells for 15 min at room temperature in the dark condition and then detected by the Olympus Fluoview FV1000 laser scanning confocal system.
Fluoview fv1000 laser
Manufactured by Olympus
Sourced in Japan
The FluoView FV1000 is a laser scanning confocal microscope system designed for high-resolution fluorescence imaging. It offers advanced optics and a modular design to enable customization for a variety of imaging applications.
Automatically generated - may contain errors
7 protocols using fluoview fv1000 laser
1
Apoptosis Detection by Annexin V-FITC/PI
2
Caspase-3/9 Assay Kit Protocol
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All processes were instructed following the protocol of caspase-3/9 assay kit (Biovision, Milpitas, CA). Olympus Fluoview FV1000 laser scanning confocal system (Olympus America Inc., Center Valley, PA) at 400 nm excitation and 505 nm emission was employed here for photosensitive reaction measurement. The fold of changes was calculated relative to the untreated cells.
3
Overexpression of NtDREB-1BL1 in Tobacco
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The NtDREB-1BL1 gene was amplified by PCR from the cDNA of N. tabacum leaves with the primers (Table S1 ). The overexpression vector of Sp1300-NtDREB-1BL1-GFP was constructed by XbaI and KpnI restriction enzymes, which was constructed to express NtDREB-1BL1 with a GFP-tag at the C terminus under the control of the cauliflower mosaic virus 35S promoter (Table S1 ). The recombinant vectors were transferred into Agrobacterium (GV3101) by electroporation and cultured at 30 °C for 2 days. The positive Agrobacterium was scraped from the solid dish with the inoculation loop and cultured in 10 mL YEB medium. Agrobacterium cells were suspended with 10 mM MgCl2 (including 120 μM acetosyringone, AS) and adjusted OD600 to 0.6. N. benthamiana plants with about five leaves were infected with Agrobacterium for the transient expression of NtDREB-1BL1-GFP protein. Fluorescence observation was performed by a FluoView FV 1000 laser scanning confocal microscopy system (Olympus, Tokyo, Japan) as described previously [31 (link)].
4
Confocal Imaging and Fluctuation Analysis
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Confocal images and fluorescence fluctuation data were acquired with an Olympus FluoView FV1000 laser scanning microscope (Olympus Corporation, Center Valley, PA) and a PlanApo N 60X 1.42 NA oil-immersion objective (Olympus) using 488 nm excitation for mEGFP, BODIPY-FL, and Alexa-488, and 543 nm excitation for mCherry. The primary dichroic filter for excitation was 405/488/559/635, and for emission, the band pass filters used were 505–525 nm and 560–660 nm for the green and red channels, respectively. The pinhole was set at 95–105 μm. Lasers were set at 1% output (using the software control slider) unless otherwise stated. Detectors were set to the pseudo-photon counting mode. High-resolution images of cells were acquired at 1,024 × 1,024 pixels using Kallman averaging every two lines. Cell culture dishes were mounted on a heated stage set at 37 °C and imaged for < 4 h.
5
Confocal and Wide-field Microscopy for Imaging Actin Dynamics
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Confocal microscopy was performed on a FluoView FV1000 laser scanning confocal microscope (Olympus, PA) equipped with a 60x magnification water-immersion objective, 100-W halogen illumination, and a multiline 488-nm-wavelength argon laser. Fluorescent light and transmitted light were collected simultaneously in z stacks with 1-µm increments along the z axis. For imaging of phalloidin-stained actin, the pinhole was opened almost completely and single z sections were captured. For imaging of transfected cells, z stacks were captured, a single slice of the red and DIC channels was presented, and the entire z stack from the green channel was compiled into a maximum-intensity projection before making the composite image. Wide-field microscopy was performed on an Olympus IX-70 inverted microscope for bright-field and phase-contrast imaging of live blebbing cells in real time (30 frames per second [fps]) with a QImaging QI Click charge-coupled-device (CCD) camera and Volocity software.
6
Imaging Anesthetized Embryonic Vasculature
7
Immunofluorescence Labeling of AFP Fragments
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Firstly, cells were diluted with DMEM (Dulbecco's Modified Eagle Medium) without serum into a suspension of 2 × 104 cells/ml, and 500 μl suspension cells were added into a chamber slide. Then add 30 μl FITC‐AFP390–609 fragment (FITC‐AFP390–609 fragment link used HOOKDye Labeling Kit). Samples were culture at 37°C avoid light, with 5% CO2 for 37 h. Secondly, the cells were washed three times (5 min each) with PBS, added 500 μl 4% paraformaldehyde‐fixed cell 30 min and used PBS wash three times. Thirdly, added 200 μl DAPI for 10 min, wash with PBS for three times, then, added 50 μl fluorescent quencher and observed with an Olympus FluoView FV1000 laser.
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