The intracellular ROS levels were determined using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) as indicator, which was non-fluorescent and could be oxidized to generate fluorescence. MCF-7 cells (5 × 104) were seeded in single-well plates with glass bottom. After 24 h incubation, the culture medium was replaced with 1 mL of culture medium containing NPs at a concentration of 500 μg/mL and the cells were incubated for another 2 h and 6 h. Then, the medium was removed and the cells were rinsed with PBS for three times. After staining with DCFH-DA (10 μM, 30 min) and Hoechst 33,342 (10 μg/mL, 15 min), the cells were fixed with 4% paraformaldehyde (30 min) and imaged using a laser confocal microscope (Delta Vision TM Elite, GE).
Deltavision elite
Manufactured by GE Healthcare
Sourced in United States
The DeltaVision Elite is a microscope system designed for high-resolution imaging of cellular structures and dynamics. It utilizes advanced optical technology to provide clear and detailed images of samples. The core function of the DeltaVision Elite is to enable researchers to observe and analyze cellular processes with exceptional resolution and precision.
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Lab products found in correlation
Softworx, by GE Healthcare (9 mentions)
Hoechst 33342, by Merck Group (5 mentions)
Softworx software, by GE Healthcare (4 mentions)
Alexa fluor 488 goat anti rat, by Thermo Fisher Scientific (4 mentions)
Matlab, by MathWorks (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Alexa 488, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Fm4 64, by Thermo Fisher Scientific (3 mentions)
Mitotracker red, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Deltavision image restoration microscope, by GE Healthcare (2 mentions)
Leibovitz s l 15, by Thermo Fisher Scientific (2 mentions)
Bx81 microscope, by Olympus (2 mentions)
Oil uplsapo100xo objective, by Hamamatsu Photonics (2 mentions)
Cellm software package, by Olympus (2 mentions)
Deltavision elite microscope, by GE Healthcare (2 mentions)
Orca er camera, by Hamamatsu Photonics (2 mentions)
Coolsnap hq2, by GE Healthcare (2 mentions)
1.4 na objective, by Olympus (2 mentions)
137 protocols using deltavision elite
1
Intracellular ROS Measurement in MCF-7 Cells
2
Cellular Uptake of Micelle-Encapsulated Doxorubicin
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To evaluate the cellular uptake of TW80-Fe3O4-ART micelles, MCF-7 cells were cultured in glass-bottom cell culture dishes for 24 h. Subsequently, the cells were exposed to both free DOX and DOX-labeled TW80-Fe3O4-ART micelles at equivalent DOX concentrations (10 µg/mL) for either 1 or 4 h. Cytoplasmic and nuclear staining was performed using Lysogreen (2 μM) and Hoechst 33342 (10 μg/mL), respectively. Following a single wash with PBS, the uptake patterns were visualized using confocal laser scanning microscopy (Delta Vision TM Elite, General Electric, Boston, MA, USA).
3
Cellular Uptake Analysis of PHD@MnO2
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HMME exhibits red fluorescence; thus, intracellular fluorescence can be directly observed to evaluate the cellular uptake of PHD@MnO2. MCF-7 cells (5 × 104 cells /mL) were seeded in 6-well plates and incubated at 37 °C in 5% CO2 for 24 h. PHD@MnO2 (equivalent to 10 μg/mL HMME) was suspended in 1 mL of fresh medium and the cells were further incubated. The medium was removed at 0.5, 1, 2, 3, and 4 h, and the cells were washed with PBS three times. LysoGreen (1 mL) was added, and the cells were incubated for 30 min to stain the lysosomes. Hoechst 33342 (1 mL, 10 μg/mL) was then added to cells and incubated for 15 min to stain the nuclei. After washing with PBS, the cells were fixed with 4% paraformaldehyde. The cells were then observed using a laser confocal microscope (Delta Vision TM Elite, General Electric Company, USA).
4
Intracellular ROS Generation Evaluation
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To investigate intracellular ROS generation, 2,7-dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescent probe. MCF-7 cells were seeded in a six-well plate (5×104 cells/well) and incubated for 24 h. Next, the medium was replaced with 1 mL medium containing HMME, PH nanoparticles, or PHD@MnO2 nanoparticles (equivalent to 15 μg/mL HMME) and incubated for 4 h. The spent medium was then removed, and the cells were rinsed with PBS three times. After incubation with 10 μM DCFH-DA for another 30 min, serum-free medium was added, and some groups subsequently received ultrasound treatment for 180 s (1 MHz, 1.75 W/cm2). The spent medium was then replaced with 1 mL Hoechst 33342 (10 μg/mL), and the cells were incubated for 15 min. Afterward, the cells were fixed with 4% paraformaldehyde for 30 min. Fluorescence images were taken under a laser confocal microscope (Delta Vision TM Elite, General Electric Company). The mean optical density of the green fluorescence representing the ROS levels in different groups was quantified using ImageJ software.
5
Immunofluorescence Staining and Imaging
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Cells grown on coverslips were fixed in 4% paraformaldehyde in PBS for 10 min followed by permeabilization with 0.5% Triton X-100 for 5 min for DAPI (Sigma) staining and mounted in gelvatol. Slides were imaged on a DeltaVision Image Restoration Microscope with a ×100 objective (DeltaVision Elite, GE) (Li and Noegel, 2015 (link)).
6
Immunofluorescence Staining of β-catenin and COPB
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Cells were fixed, washed, blocked and incubated with primary antibody (the same anti-β-catenin or anti-COPB antibody used for Western blot and IP). Cells were washed and secondary antibody/Hoechst nuclear stain added. Cells were washed and slides mounted using Vectashield. The slides were imaged using the DeltaVision Elite (GE Healthcare) at a magnification of x100 and SoftWoRx Explorer 1.3 (GE Healthcare) was used for analysis. Cells requiring a CSK ( 100 mM NaCl, 300 mM sucrose, 1 mM MgCl2, 10 mM PIPES, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 0.1% Triton X-100 and PI) wash were washed on ice before fixation for 1 min.
7
Live Cell Photobleaching Analysis
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Live cells expressing GFP constructs of interest were seeded in 35mm glass-bottom plates and visualized on the DeltaVision Elite (GE Healthcare) in a 37C and 5% CO2 environmental chamber using a 60× Silicone Plan-Apo/1.3 NA objective. The system was controlled by the Elite Master Workstation for bleaching and image acquisition. Photobleaching was performed with a 1 s 10% laser (488nm) power, 10 mW bleachpoint. For FLIP, the bleach point was subject to continuous bleaching at 6 s intervals for a total of 180 s. For FRAP, five pre-bleach measurements were taken before photobleaching and images were taken at 3.5 s intervals for a total of 180 s during recovery. Region intensity measurements were made in ImageJ and normalized as previously described in (Mekhail et al., 2005 (link)). All photobleaching graphs represent the average of at least 5 cells.
8
Z-stack imaging via Deltavision Elite
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Image acquisition: For each timepoint, Z-stacks were acquired on a Deltavision Elite (GE Life Sciences). Samples were illuminated through a 60x/1.42NA objective lens with excitation light passing through a bandpass filter of 475/28. Fluorescence emission was collected through a bandpass filter of 525/48 and sampled on an Edge 5.5 sCMOS camera (PCO) at 108nm and 200nm in the lateral and axial axes respectively. To minimize spherical aberration, the immersion oil refractive index was matched until point spread functions from within the sample itself were approximately symmetrical as described by Hiraoka et al. Biophys, 1990. Z-stacks were deconvolved using the constrained iterative algorithm with an appropriately matched optical transfer function in SoftWorx (GE Healthcare).
9
High-Resolution Live-Cell Imaging Protocol
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All imaging was conducted on an DeltaVision Elite (GE Healthcare) equipped with a Plan Apochromat 60× 1.40 NA oil objective lens and a Plan Apochromat 60× 1.49 NA total internal reflection microscopy (TIRF) oil objective lens (Olympus) and a 488-nm laser from DeltaVision Quantifiable Laser Module, and images were captured with a pco.edge sCMOS camera with near-perfect linearity across its 15-bit dynamic range. The pixel size of the sCMOS camera was 0.1077 µm. The entire microscope was housed within a temperature-controlled environmental chamber. All image acquisition was done in SoftWoRx (6.0; GE Healthcare). Files were imported into ImageJ (National Institutes of Health) and analyzed as described in the following sections. No gamma adjustment was applied during figure preparation.
10
TIRF Microscopy of Pollen Tube Dynamics
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For TIRF microscopy of PTs, a very planar gel pad was generated by laying two microscope slides orthogonal on the edges of three adjacent slides (Figure S3 ). 500 μl of molten PGM containing 2% agarose was pipetted to the middle of the lower slides and immediately covered with another slide. After solidification, the uppermost slide and all flanking slides were removed and the PGM pad was hand pollinated as described above (Pollen Mounting and Live Cell Imaging). Pollinated slides were kept in a damp box for 3–5 h. Prior to microscopy, a droplet of double distilled water was pipetted onto the pad and a No. 1.5H cover slip was added. TIRF illumination was generated in a Delta Vision Elite (GE, Healthcare, Applied Precision) system with an Olympus IX-71 microscope, equipped with an Insight SSI(TM) solid state illumination system and an X4 laser module. Images were taken with an Olympus UAPON 100XOTIRF 1.49 NA oil immersion objective and recorded with a CoolSnap HQ2 CCD camera (Photometrics, Tucson, USA). GFP was excited with the 488 nm laser line and emission was detected between 501 and 549 nm. Image exposure time and TIRF angle were adjusted according to sample fluorescence intensity and specimen location.
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