To detect the effect of nanofibers on scavenging intracellular ROS, DCFH-DA staining (Beyotime, China) was used. Briefly, after RAW 264.7 cells cocultured with LPS and nanofibers for 24 h, DCFH-DA was used to incubate the samples for 30 min, then the live RAW 264.7 cells were stained by using Hoechst 33342 solution (Beyotime, China). For the blank group, RAW 264.7 cells were added to the plates. The results were detected by laser scanning confocal microscope at 485 nm.
Dcfh da staining
Manufactured by Beyotime
Sourced in China
DCFH-DA staining is a fluorometric assay used to detect the presence of reactive oxygen species (ROS) within cells. DCFH-DA (2',7'-Dichlorodihydrofluorescein diacetate) is a cell-permeable compound that can be oxidized by ROS to produce a fluorescent signal. The intensity of the fluorescence is proportional to the level of ROS present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342 solution, by Beyotime (1 mentions)
Dcfh da, by Beyotime (1 mentions)
Confocal microscopy, by Nikon (1 mentions)
Cck 8 assay kit, by Beyotime (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Standard assay kits, by Beyotime (1 mentions)
Mitosox staining, by Thermo Fisher Scientific (1 mentions)
Salubrinal, by Merck Group (1 mentions)
Diphenyleneiodonium dpi, by Merck Group (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab59348, by Abcam (1 mentions)
Jc 1 staining, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc pi, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Tunicamycin, by Merck Group (1 mentions)
Dl homocysteine, by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Beyotime (1 mentions)
Axio observer d1, by Zeiss (1 mentions)
8 protocols using dcfh da staining
1
Nanofiber-Mediated ROS Scavenging in Macrophages
2
Measuring Oxidative Stress in PC12 Cells
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ROS generation was measured via DCFH-DA staining to detect intracellular hydrogen peroxide and oxidative stress (Beyotime, Shanghai, China). In brief, PC12 cells in 6-well plates were treated via Aβ1-42, Fer-1 or si-VDAC1/si-NT. Then, 10 μM DCFH-DA was added into cells and reacted for 15 min. After washing twice with DMEM, the cells were examined via confocal microscopy (Nikon, Japan).
3
Measurement of Cellular Oxidative Stress
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Cell ROS production was evaluated by DCFH-DA staining (Beyotime, Shanghai, China). Briefly, H9c2 cells were stained with DCFH-DA (5.0 μmol/L) in the dark at 37 °C for 30 min and were then visualized in a blinded manner using an Olympus fluorescence microscope (Olympus, Tokyo, Japan). Cell viability was determined using the CCK-8 assay kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol.
4
Apoptosis and Oxidative Stress Quantification
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TUNEL labeling (Roche, Shanghai, China) and caspase-3 activity colorimetric assay kits (KeyGEN Biotech, Jiangsu, China) were used to determine cellular apoptosis as described previously [18 (link)]. The apoptosis index was obtained by dividing the number of TUNEL-positive apoptotic cells by the total number of nucleated cells stained with 4′,6-diamino-2-phenylindole (DAPI). The caspase 3 colorimetric assay kit is based on the hydrolysis of acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase 3, resulting in the release of the p-nitroaniline (pNA) moiety. The p-NA produces a yellow color and can be quantified using a spectrophotometer or a microtiter plate reader reading absorbance at 405 nm. DCFH-DA staining (Beyotime Biotechnology, Nantong, China) and MitoSOX staining (Invitrogen, Carlsbad, USA) were used to measure cellular and mitochondrial ROS levels in the cardiomyocytes. DCFH was added to the cardiac homogenate to evaluate ROS production, and the fluorescence was measured using a fluorometer [19 (link)]. Total superoxide dismutase (SOD), cellular CuZnSOD, mitochondrial MnSOD, and malondialdehyde (MDA) levels were determined utilizing standard assay kits (Beyotime Biotechnology, Nantong, China) following the manufacturer's recommendations.
5
Homocysteine-Induced HUVEC Apoptosis
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Human umbilical vein endothelial cells (HUVEC) were obtained from the Cell Line Bank of the Chinese Academy of Sciences (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco Technologies (Logan, UT, USA). DL-Homocysteine, tunicamycin, diphenyleneiodonium (DPI), and salubrinal were obtained from Sigma (St Louis, MO, USA). siRNA and Lipofectamine® 2000 were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against PERK (3192s), p-PERK (3179s), eIF2a (5324s), p-eIF2a (3597 s), ATF4 (11815S), caspase-3 (9662), cytochrome c (4272), and actin (12262s) were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against GRP78 (ab21685), CHOP (ab11419), p-eNOS (76199), Bax (ab32503), and Bcl-2 (ab59348) were purchased from Abcam (Boston, MA, USA). Electrochemiluminescence (ECL) Western blotting detection reagents were obtained from Millipore (Bedford, MA, USA). Cell Counting Kit-8 (CCK-8) and DCFH-DA staining were purchased from Beyotime (Jiangsu, China). JC-1 staining, mitochondrial ROS fluorescent probe, and Annexin V-FITC/PI were obtained from Molecular Probes (Invitrogen, Milan, Italy).
6
Quantifying Oxidative Stress via ROS
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ROS generation was detected by DCFH-DA staining (Beyotime Biotechnology, China). After washing with PBS, the cells were incubated with DCFH-DA (10 μmol/L) at 37°C in the dark for 30 min. Fluorescent images were captured with a fluorescence microscope (ZEISS Axio Observer D1, Germany) in a blinded manner. To further assess the oxidative stress level, the content of malondialdehyde (MDA) was measured by a commercially available kit (Beyotime Biotechnology, China).
7
Quantifying Cellular ROS Levels
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DCFH-DA staining (Beyotime, China) was used to determine ROS. Briefly, the harvested cells were stained with 2.5 μM DCFH-DA for 30 min, and washed twice with PBS. After washing, 1 × 105 cells were quantified by samples, and ROS level was measured by flow cytometry (BD, USA). Cell Number was chosen according to manufacturer's instructions.
8
Intracellular ROS Measurement by DCFH-DA
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Generation of intracellular ROS was measured by using DCFH‐DA staining (Beyotime). In brief, cells were plated at a density of 2.5 × 105/well in six‐well plates and exposed to vehicle or alternol (2.5 and 5.0 μM) for 12 hrs. Then, the cells were trypsinized and incubated with DCFH‐DA (10 μM) for 30 min. at 37°C in dark. Reactive oxygen species generation was determined by confocal microscopy and flow cytometer.
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