A dichloro-dihydro-fluorescein diacetate (DCFH-DA) detection kit was used to assess the ROS level in HUVECs. After pretreatment with the 5 µg/mL of Mv, Mv-3-glc, Mv-3-gal, or BAE for 24 h and continuing with 5.5 mM or 30 mM glucose for 24 h, cells were washed with PBS, and then 10 µM DCFH-DA was added to each well and reacted for 20 min at 37 °C, and the cells were washed thoroughly with PBS. A group of cells was visualized under an IX53 inverted fluorescent microscope (Olympus, Tokyo, Japan) with 530 nm emission and 485 nm excitation filters immediately. All images presented are in ×200 magnification. Another group of cells was collected after dissociation, and fluorescence was recorded by a Synergy H4 multi-mode microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The total fluorescence intensity of cells in each well was noted, and ROS generation was measured as a fold of the control.
Ix53 inverted fluorescent microscope
Manufactured by Olympus
Sourced in Japan
The IX53 is an inverted fluorescent microscope designed for biological and life science applications. It features optical components and illumination systems optimized for fluorescence imaging. The IX53 enables researchers to observe and analyze samples using fluorescence techniques.
Automatically generated - may contain errors
Lab products found in correlation
Cellsens software, by Olympus (2 mentions)
Fluoview fv1200 confocal laser scanning microscope, by Olympus (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Synergy h4 multi mode microplate reader, by Agilent Technologies (1 mentions)
Synergy h4, by Agilent Technologies (1 mentions)
Ma5 13188, by Thermo Fisher Scientific (1 mentions)
D1396, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Alginate lyase, by Merck Group (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Dcfh da detection kit, by Beyotime (1 mentions)
Tristar lb 941 microplate reader, by Berthold Technologies (1 mentions)
Bs 0295g fitc, by Bioss Antibodies (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by RiboBio (1 mentions)
Cell counting kit 8, by Beyotime (1 mentions)
10 protocols using ix53 inverted fluorescent microscope
1
ROS Quantification in HUVECs
2
ROS Measurement in HepG2 Cells
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The reactive oxygen species (ROS) in HepG2 cells were assessed using the DCFH-DA detection kit. After an initial treatment with the 5 μg/mL of Mv, Mv-3-glc, Mv-3-gal, or BAE for 24 h which was later followed by a 5 mM or 30 mM glucose treatment for 24 h, the cells were washed with PBS, and then 10 μM DCFH-DA was added to each well and allowed to react for 20 min at 37 °C. The cells were again washed thoroughly with PBS and then a group of these cells was immediately observed under an IX53 inverted fluorescent microscope (Olympus, Tokyo, Japan) at 530 nm emission and 485 nm excitation filters. The images are presented under 200× magnification. After dissociation, another group of cells was collected and their fluorescence was recorded by a Synergy H4 multi-mode microplate reader (BioTek Instruments Inc., Winooski, VT, USA). The total fluorescence intensity of cells in each well was noted, and ROS generation was measured as a fold of the control.
3
Visualizing Angiogenesis in HUVEC Scaffold
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To observe migration, morphology, and angiogenesis of the embedded HUVEC, F-actin, CD31, and nuclei were stained using rhodamine-phalloidin, anti-CD31, and DAPI, respectively. First, the formulated scaffold was fixed with 4% paraformaldehyde (P6148, Sigma-Aldrich, U.S.A.) for 40 min at room temperature (RT). The fixed scaffold was immersed in alginate lyase (Sigma-Aldrich, U.S.A.) solution to remove alginate at 37°C. The alginate removed HUVEC core was immersed in a collagen matrix and incubated at 37°C for gelation. Subsequently, the HUVEC core in collagen was permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, U.S.A.) for 5 min at RT. Primary antibody of anti-CD31 (MA5-13188, Invitrogen, U.S.A.) was incubated at 4°C overnight. Then, secondary antibodies (Alexa Fluor 488, Invitrogen, U.S.A.) and Phalloidin (Alexa Fluor 488, Invitrogen, U.S.A.) were applied for 2 h at RT. Besides, nuclei of the HUVEC core were stained with DAPI (D1396, Invitrogen, U.S.A.) for 5 min. After every chemical treating step, the treated sample was washed 3 times with PBS for 5 min. The stained samples were observed using an IX53 inverted fluorescent microscope (Olympus, Japan) and a FV1000 laser scanning confocal microscope (Olympus, Japan).
4
Fluorescent Microscopy of Cell-Seeded Scaffolds
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The cell-stained scaffolds were observed using an IX53 inverted fluorescent microscope (Olympus, Tokyo, Japan) and the images were captured using the CellSens software (Olympus, Tokyo, Japan). For three-dimensional imaging, the FLUOVIEW FV1200 laser scanning confocal microscope (Olympus, Tokyo, Japan) was utilized. The live/dead cell images were analyzed using the ImageJ 1.51h software (National Institutes of Health, Bethesda, MD, USA).
5
Apoptotic Nuclear Condensation Analysis
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HSC‐T6 cells were stained with 10 μmol/L Hoechst 33258 dye for 15 min. The stained cells were imaged using an IX53 Inverted Fluorescent Microscope (Olympus) with 460 nm emission and 350 nm excitation filters. The nuclear DNA was blue fluorescent. The number of cells with apoptotic nuclear condensation was counted in each well. The TEM was performed following the method previously reported (Lee & Friedman, 2011 ) by a FEI Tecnai G2 Spirit Bio TWIN Microscopes Electron Microscopy (FEI).
6
Measuring Oxidative Stress in HSC-T6 Cells
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The HSC‐T6 cell's ROS levels were measured by DCFH‐DA detection kit (Beyotime). Briefly, after Mv treatment (final concentrations: 0, 50, 75, and 100 μg/ml, respectively) for 24 hr, HSC‐T6 cells were stained with 10 μM/ml of H2DCFDA at 37°C for 20 min in the dark. The relative ROS levels in the HSC‐T6 cells were observed by an IX53 Inverted Fluorescent Microscope (Olympus). Fluorescent intensity was measured at the 530 nm emission wavelength and 485 nm excitation wavelength, respectively.
7
Quantifying Oxidative Stress in Endothelial Cells
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Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay is a quantitative method for oxidative stress assessment in cells. In this study, DCFH-DA detection kit was used to assess the ROS level in endothelial cells. Briefly, the cells were seeded in 6-well plates, treated with different samples to incubate for 24 h. After washing cells with PBS, 10 μmol/L DCFH-DA was added to each well and reacted for 20 min at 37°C, and then the cells were washed thoroughly with PBS. A group of cells was visualized under an IX53 Inverted Fluorescent Microscope (Olympus, Tokyo, Japan) at 530 nm emission and 485 nm excitation filters immediately. All images presented are in ×200 magnification. Another one was collected in 1 mL PBS after dissociated, and fluorescence was recorded by LB 941 TriStar Microplate Reader (Berthold Technologies, Bad Wildbad, Germany). The total fluorescence intensity of cells in each well was noted, and ROS generation was measured as fold increase over the untreated control.
8
Fluorescence Imaging of Cell Scaffolds
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The cell-stained scaffolds were observed under the IX53 inverted fluorescent microscope (Olympus, Japan) and the images were captured using the CellSens software (Olympus, Japan). For threedimensional imaging, the FLUOVIEW FV1200 laser scanning confocal microscope (Olympus, Japan) was utilised. The live/dead cell images, the cell-cell contacts images, and the newly-produced type-I collagen matrix images of the cell scaffolds were analysed using the ImageJ 1.51h software (National Institutes of Health, U.S.A.).
9
Immunofluorescence Analysis of Myoblast Differentiation
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For immunofluorescence analysis, C2C12 myoblasts were transfected with miR-23a-5p mimics, NC, si-lncDum, p-lncDum, si-NC or pcDNA3.1+ empty vector during differentiation, then washed three times with PBS, and fixed in 4% paraformaldehyde for 30 min at room temperature. Next, cells were washed three times with PBS, and blocked with 5% normal goat serum in PBS for 1 h at room temperature. Then, cells were incubated with an anti-myosin slow antibody (anti-MyHC1; dilution 1:300; cat. no. bs-9862R; BIOSS) overnight at 4°C, washed three times with PBS, and incubated with a fluorescent secondary antibody (dilution ratio 1:300; cat. no. bs-0295G-FITC; BIOSS) at 37°C for 1 h. Cell nuclei were stained with DAPI (Beyotime Institute of Biotechnology) for 20 min at room temperature. Images (magnification, ×100) were captured using an IX53 fluorescent inverted microscope (Olympus Corporation). The fusion index of myotubes as calculated by Image J software (Image J 1.50i; National Institutes of Health).
10
Regulation of C2C12 myoblast proliferation by miR-23a-5p and lncDum
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In brief, C2C12 myoblasts were seeded in 96-well plates, and transfected with miR-23a-5p mimics, NC mimics, si-lncDum, p-lncDum, si-NC or pcDNA3.1+ empty vector, after which cell proliferation was determined at 0, 12, 24, 48, 72 and 96 h using a Cell Counting Kit (CCK)-8 (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. For 5-ethynyl-2′-deoxyuridine (EdU) proliferation analysis, C2C12 myoblasts were treated with 10 µM EdU (Guangzhou RiboBio Co., Ltd.) after 24 h post-transfection and incubated for 3 h at 37°C. EdU staining was performed according to the manufacturer's protocol. Cell nuclei were stained with DAPI (Beyotime Institute of Biotechnology) for 10 min at room temperature. Images (magnification, ×100) of the entire well were captured using an IX53 fluorescent inverted microscope (Olympus Corporation).
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