All reagents and solvents used in the experiments were commercially available and used without further purification. PBS buffer (1X, pH 7.2–7.4, Adamas life, China). DCFH-DA (2’,7’-Dichlorofluorescein diacetate, Beyotime Biotechnology, China). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-um bromide), Annexin-V-FITC and propidium iodide (PI) (Solarbio, Beijing, China). Mitochondrial membrane potential assay kit with JC-1 (Beyotime Biotechnology, Shanghai, China). Gallium(III) nitrate nonahydrate (Ga(NO3)3·9H2O, Adamas, Shanghai, China). 5-Bromo-8-hydroxyquinoline (Adamas, Shanghai, China). Cisplatin (Macklin, Shanghai, China). Oxaliplatin (Leyan, Shanghai, China). Ultrapure water (18 M Ω·cm resistivity from an EASY Ultra-pure Water System, Heal Force, Shanghai, China) was used in the experiments. MCE membrane filters (0.22 μm, Jinteng, Tianjin, China) for suction filtration were used. The microwave synthesizer (SP, UWave-2000, SINEO, Shanghai, China) for the reaction was used.
2 7 dichlorofluorescein diacetate dcfh da
Manufactured by Beyotime
Sourced in China, United States
2',7'-dichlorofluorescein diacetate (DCFH-DA) is a cell-permeable fluorogenic probe. It is used to detect the presence of reactive oxygen species (ROS) in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
N acetylcysteine nac, by Beyotime (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Hydrogen peroxide, by Tianjin Damao (3 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
P akt ser473, by Cell Signaling Technology (2 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Sartorius (2 mentions)
Propidium iodide, by Beyotime (2 mentions)
Rosup, by Beyotime (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Bupivacaine hydrochloride, by Merck Group (2 mentions)
Anti β actin, by Kangchen Biotech (2 mentions)
Glucose, by Merck Group (2 mentions)
83 protocols using 2 7 dichlorofluorescein diacetate dcfh da
1
Gallium-based Anticancer Therapy Development
2
Prion Protein Analysis Techniques
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Three dyes, ThT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DAPI, and two antibodies, the mouse anti-PrP monoclonal antibody 3F441 (link),48 (link) and a 10-nm gold-labeled anti-mouse antibody7 ,9 (link),57 (link), were purchased from Sigma-Aldrich (St. Louis, MO). Sarkosyl and PK were obtained from Amresco (Solon, OH). Alexa 546-conjugated fluorescent secondary antibody7 ,9 (link),57 (link), the proteasome inhibitor MG132, and the ROS probe DCFH-DA (2′,7′-dichlorofluorescein diacetate) were purchased from Beyotime (Nantong, China), respectively. Ni-Sepharose was obtained from GE Company (Pittsburgh, PA). All other chemicals used in this study were of analytical grade and were produced in China.
3
Asiaticoside Modulates PI3K/Akt/mTOR Signaling
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Asiaticoside was purchased from Selleck Chemicals (Houston, TX, USA), Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Biological Industries (BI, Israel). Penicillin/streptomycin and trypsin were purchased from Corning Incorporated (Corning, NY, USA). The chemotherapy drugs paclitaxel (PTX), Adriamycin (ADM), colchicine, and vincristine were purchased from Energy Chemicals (Shanghai, China). The primary monoclonal antibodies of PI3K-p110α (#4255), PI3K-p110β (#3011), PI3K-p110γ (#4252), p-PDK1 (Ser241) (#3061), p-Akt (Ser473) (#4060), p-mTOR (Ser2448) (#2976), p-ERK1/2 (#4370), ERK1/2 (#4696), p-JNK1/2 (#9255), JNK1/2 (#9252), p-P38 (#4511), P38 (#8690), β-actin (#4970), and P-gp (ABCB1) (#13342) were purchased from Cell Signaling Technology (Danvers, MA, USA). HRP-conjugated secondary goat anti-mouse antibody, Annexin V-FITC, propidium iodide (PI), and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Beyotime Biotechnology (Nantong, JS, China). All other chemicals were purchased from Sigma Aldrich (MO, USA).
4
Antioxidant and Anti-inflammatory Effects
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Active baker's Yeast was purchased from Angel Yeast Co., Ltd. (Yichang, China). Curcumin and EGCG were purchased from Macklin (Shanghai, China). DSS was purchased from Yeasen (Shanghai, China). Phosphate buffer saline (PBS) was purchased from Sevier (Wuhan, China). 2,7-dichlorofluorescein diacetate (DCFH-DA) was purchased from Beyotime Biotechnology Co., Ltd. Fluorescent lipophilic dyes (DiL and DiR) were purchased from Promokine (Heidelberg, Germany). MTT, myeloperoxidase (MPO) assay kit, lipopolysaccharides (LPS), and antibodies (CD86, CD80, and CD206) were purchased from Meilun Bio (Dalian, China).
5
Measuring Intracellular ROS Levels
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The ROS levels in cells were measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Beyotime). N-acetylcysteine (NAC) (Beyotime), a ROS inhibitor, was pre-treated before LPS administration. Cells were treated with LPS (1 μg/mL) for 24 h. Then, the cells were washed twice with cold PBS, treated with DCFH-DA (10 μM final concentration), and incubated for 30 min at 37°C in a light-protected, humidified chamber. After probe treatment, the cells were washed at least twice with cold PBS. A fluorescence microscope (Nikon) was used at set excitation and emission wavelengths in these experiments.
6
ROS Measurement in HEK293T Cells
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For ROS measurement experiments, 1 × 106 HEK293T cells were seeded onto 6‐well plates and transfected at ≈70% confluence with 2 µg of each DdCBE/TALED monomer to make up 4 µg of total plasmid DNA using PEI transfection reagent. Changes in the intracellular level of ROS were determined using 2′, 7′‐dichlorofluorescein diacetate (DCFH‐DA) (Beyotime, China). Culture medium was first removed and the cells were washed three times with PBS. DCFH‐DA, diluted to a final concentration of 10 µm with DMEM, was added to cultures and incubated for 20 min at 37 °C. Then DCF fluorescence distribution of 1 × 106 cells was monitored with excitation wavelength at 488 nm and emission wavelength at 525 nm. The increase value compared to control (untreated or Dead‐DdCBE treated cells) was viewed as the increase of intracellular ROS.
7
Exploring the Anticancer Potential of TSG
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2,3,5,4′-Tetrahydroxystilbene glucoside (TSG), purity >98%, was purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Sodium azide (NaN3) was obtained from Ameresco (USA). Antibodies against Bcl-2, Bax, β-actin and horseradish peroxidase-conjugated goat anti-rabbit IgG were from Zhongshan Goldenbridge Biotechnology Co., Ltd. (Beijing, China). Annexin V/PI detection apoptotic kit, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi- dazolylcarbocyanine iodide (JC-1) were purchased from Beyotime Institute of Biotechnology (Jiangsu, China).
8
Multiparametric Flow Cytometry Analysis
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Flou-3-pentaacetoxymethyl (Fluo-3/AM; Beyotime) was used for intracellular Ca2+ testing. K562 cells from each group were collected and resuspended with 1 μmol/L Fluo-3/AM diluted in 1 mL D-Hank's balanced salt solution (Solarbio, Beijing, China) for 30 min. Then, the cells were incubated for another 30 min and analyzed by a C6 flow cytometer (BD Biosciences).
A 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) kit (Beyotime) was used for MMP measurement. K562 cells at 24 h after coculturing were stained with 0.5 mL JC-1 solution. All samples were incubated for 20 min and detected by a C6 flow cytometer.
2′,7′-Dichlorofluorescein diacetate (DCFH-DA, Beyotime) was used for ROS determination. Twenty-four hours after coculturing, K562 cells were stained with 10 μL of DCFH-DA for 20 min and resuspended in D-Hank's solution, and 2′,7′-Dichlorofluorescein (DCF) fluorescence was also detected by a C6 flow cytometer.
A 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-1) kit (Beyotime) was used for MMP measurement. K562 cells at 24 h after coculturing were stained with 0.5 mL JC-1 solution. All samples were incubated for 20 min and detected by a C6 flow cytometer.
2′,7′-Dichlorofluorescein diacetate (DCFH-DA, Beyotime) was used for ROS determination. Twenty-four hours after coculturing, K562 cells were stained with 10 μL of DCFH-DA for 20 min and resuspended in D-Hank's solution, and 2′,7′-Dichlorofluorescein (DCF) fluorescence was also detected by a C6 flow cytometer.
9
ROS Modulation and Quantification
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2 × 104 cells were seeded into a 96‐well plate; an ROS inducer, Rosup (Beyotime, Nantong, China; Cat. # S0033S‐2), was used as the positive control, and N‐acetylcysteine (NAC; MedChenExpress, Princeton, NJ, USA; Cat. # HY‐B0215) was used as an ROS inhibitor. The NAC was incubated 24 h ahead of schedule. After 2 h of DM‐AKG (Shanghai Yuanye Bio‐Technology, Shanghai, China; Cat. # S30041) administration, the serum‐free medium containing 0.1% 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA; Beyotime, Nantong, China; Cat. # S0033S‐1) was replaced. Following incubation at 37 °C for 20 min, the cells were washed three times with the serum‐free medium and were then screened and analysed with Operetta (PerkinElmer, Waltham, MA, USA), a high content profiler.
10
Intracellular ROS Detection via DCFH-DA
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We used 2',7'-dichlorofluorescein diacetate (DCFH-DA) (Beyotime, Hangzhou, China) to estimate the intracellular generation of ROS. Cells were treated with VK3 (15 μM) for 8 or 16 h. Both two cells were washed by PBS followed with 50 μM DCFH-DA treatment. Then cells were observed by microscopy. At the same time, cells in another plate were harvested and FACScan flow cytometer has been used to measure the fluorescence intensity (Becton Dickinson).
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