Intracellular levels of ROS and GSH were measured as described previously [41 (link)]. In brief, oocytes were incubated in M2 medium supplemented with either 1 mmol/L 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) or 10 μmol/L 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (Cell-Tracker Blue) for 30 minutes at 37°C and washed with DPBS for three times afterwards. The fluorescence was measured under an epifluorescence microscope with a filter at 460 nm excitation for ROS and 370 nm excitation for GSH (DP72, Olympus, Tokyo, Japan). Finally, the fluorescence intensity of ROS and GSH was determined by using EZ-C1 Free-Viewer (Nikon, Tokyo, Japan).
Ez c1 freeviewer
Manufactured by Nikon
Sourced in Japan
The EZ-C1 FreeViewer is a software application developed by Nikon for viewing and processing images captured with Nikon's confocal microscopes. It provides basic functionalities for image visualization and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Celltracker blue, by Thermo Fisher Scientific (2 mentions)
C1 si confocal microscope, by Nikon (2 mentions)
Eclipse ti with c1 confocal system, by Nikon (2 mentions)
Fk506, by Merck Group (1 mentions)
Confocal microscope, by Nikon (1 mentions)
Cytospin system, by Thermo Fisher Scientific (1 mentions)
C1si te2000 e, by Nikon (1 mentions)
12 well chamber, by Ibidi (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Bx51 widefield microscope, by Olympus (1 mentions)
Te2000 psf inverted microscope, by Nikon (1 mentions)
D eclipse c1 confocal microscope, by Nikon (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
15 protocols using ez c1 freeviewer
1
Measuring Intracellular ROS and GSH Levels
2
Monitoring NFAT Localization in Cardiomyocytes
3
Oocyte ROS and GSH Analysis
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Denuded oocytes were added to the medium which contains 1 mmol/L 2′,7′-dichlorofluorescin diacetate (2',7'-DCFHDA) for measuring ROS or 10 μmol/L Cell Tracker Blue (Invitrogen, Carlsbad, CA, USA) for measuring GSH at 37 °C in 5% CO2 for 20 min. Then oocytes were washed by M2 three times. The fluorescence was examined under a fluorescence microscope (IX73, Olympus, Tokyo, Japan) with a filter at 460-nm excitation for ROS and 370-nm excitation for GSH. The fluorescence of each oocyte was analyzed by EZ-C1Free-Viewer (Nikon, Tokyo, Japan).
4
Immunofluorescence Assay for LC3 in Leukemia Cells
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Viable MM and leukemia cells (2.5 × 104) were spread on glass slides by centrifugation at 500 rpm (400 × g) for 5 min using a cytospin system (Thermo Fisher). Cells were fixed and staining with an anti-LC3 antibody (MBL, Nagoya, Japan) at 4°C, followed by staining with Cy3-labeled goat anti-rabbit IgG and DAPI (5 μg/ml). All samples were analyzed on a Nikon confocal microscopy (EZ-C1 FreeViewer) as described previously26 (link).
5
Intracellular ROS Detection in Bovine Oocytes
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To analyze the levels of intracellular ROS in bovine oocytes, a Reactive Oxygen Species Assay Kit was applied to detect ROS as a green fluorescent DCFH-DA signal. Oocytes of each group were incubated in 10 μmol/l DCFH-DA (in a maturation medium) for 30 min at 38.5°C. After washing three times in D-PBS containing 0.1% BSA, the samples were placed on glass dishes, and the fluorescence intensity of each oocyte was measured under a fluorescence microscope (Olympus IX73, Tokyo, Japan) and quantified using EZ-C1 Free-Viewer (Nikon).
6
Mitochondrial Function Evaluation
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This procedure was performed as described previously (59 (link)) with minor modifications. Cells were treated with 5 μm Rho 123 for 30 min at 37 °C with 5% CO2. Cells were washed with PBS and stained with Hoechst 33342. The fluorescence of Rho 123 was detected by a confocal scanning microscope (C1si-TE2000-E, Nikon, Japan). For semiquantification of fluorescence of Rho 123, four to seven microscopic fields were selected randomly, and the fluorescence values (excitation wavelength, 485 nm; emission wavelength, 535 nm) were measured using an Ez-C1 free viewer (Nikon). Concomitantly, the total number of cells stained with Hoechst 33342 (2 μg/ml) in the same field was counted, and substantial values of fluorescence of Rho 123 were normalized.
7
Biofilm Visualization and Analysis
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Biofilms were grown in 12-well chambers (ibidi GmbH, Planegg, Germany) for 24 h, gently rinsed once with water and stained either with 0.1% w/v acridine orange after fixation by incubation at 60 °C for 1 h or without fixation with the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Eugene, OR, USA), according to the manufacturer’s instructions. After removing the silicone gasket, coverslips were mounted and slides were examined by CLSM using a Nikon C1-SI confocal microscope. The image stacks collected by CSLM were analysed with the EZ-C1 Free Viewer (Nikon Corporation, Tokyo, Japan) and the Image J 1.47 (Wayne Resband, National Institutes of Health, Bethesda, MD, USA) softwares. The biofilm architecture was analysed using the COMSTAT software package [16 (link)]. Results are the mean of three independent experiments ± standard deviation.
8
Measuring Oxidative Stress in Oocytes
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The denuded oocytes were incubated in the medium containing 1 mmol/L 2′,7′-dichlorofluorescein diacetate (2′,7′-DCFHDA) for measuring ROS or 10 μmol/L CellTracker Blue (Invitrogen, Carlsbad, CA, USA) for measuring GSH for 30 min at 37 °C in 5% CO2. Then oocytes were washed with DPBS three times. The fluorescence was measured under a fluorescence microscope (DP72, Olympus, Tokyo, Japan) with a filter at 460 nm excitation for ROS and 370 nm excitation for GSH. The fluorescence of each oocyte was determined by EZ-C1Free-Viewer (Nikon, Tokyo, Japan).
9
Confocal Microscopy of Spheroids
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Spheroids were prepared for confocal microscopy as described 29 (link). Details of the antibodies used are given in Supporting Information Table S2 . Images were collected on a Nikon C1 confocal using a TE2000 PSF inverted microscope, using ×60 /NA 1.40 Plan Apo or ×20/NA 0.50 Plan Fluor objectives and ×3 confocal zoom. Different sample images detecting the same antibodies were acquired under constant acquisition settings. Images were processed using Nikon EZ-C1 FreeViewer v3.3 software. Bright-field images were collected on an Olympus BX51 widefield microscope, using a ×10/NA 0.3 UPlan F1 objective. Images were captured with a CoolSNAP camera system and processed using MetaMorph imaging v5.0 software.
10
Hypoxia Imaging in Tumor Spheroids
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Spheres were treated with pimonidazole (200 μM) for 2 h. Thereafter, spheres were centrifuged at 1,000 rpm for 5 min and fixed in 4% buffered formalin. Next, spheres were transferred in eight-well chamber slides coated with a thin layer of matrigel, permeabilized with PBS with Tween 20 (PBST; 1× PBS + 0.3% Triton X-100) followed by blocking in 5% bovine serum albumin (BSA) block buffer (PBS + 0.3% Triton X-100 + 5% BSA). Spheres were then incubated with fluorescein isothiocyanate-conjugated monoclonal antibody against pimonidazole for overnight and mounted with gold antifade reagent with DAPI. Stained spheres images were captured at 200× magnification using a Nikon D-Eclipse C1 confocal microscope and analyzed using EZ-C1 Free viewer.
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