MDA and SOD assay kits (A003-1-2 and A001-3-2 , Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) were used according to the manufacturer’s instructions to measure the MDA and SOD content, while the fluorescence dye 2’,7’-dichlorofluorescin diacetate (DCFH-DA; Beyotime Institute of Biotechnology, China) was used to determine the generation of intracellular ROS. Briefly, untreated and C3-deficient cells were plated into 12-well plates and then exposed to 3% and 5% CSE for 24 h. The DCFH-DA solution (20 μM) was added to the cells for 30 min, and the mean fluorescence intensity was measured by flow cytometry.
2 7 dichlorofluorescin diacetate dcfh da
Manufactured by Beyotime
Sourced in China
2',7'-dichlorofluorescin diacetate (DCFH-DA) is a cell-permeable compound that can be used to detect the presence of reactive oxygen species (ROS) in cells. When DCFH-DA enters the cell, it is deacetylated by cellular esterases to form 2',7'-dichlorofluorescin (DCFH), which can then be oxidized by ROS to form the fluorescent compound 2',7'-dichlorofluorescein (DCF). The intensity of the DCF fluorescence is proportional to the amount of ROS present in the cell.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Mitosox red, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Sod and mda assay kits, by Nanjing Jiancheng (2 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)
N acetyl l cysteine nac, by Beyotime (2 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (2 mentions)
Hoechst 33324, by Merck Group (2 mentions)
Hoechst 33342, by Beyotime (2 mentions)
Cisplatin, by Merck Group (2 mentions)
2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi, by Beyotime (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
Singlet oxygen sensor green (sosg), by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharide lps, by Merck Group (2 mentions)
Penicillin and streptomycin, by Merck Group (1 mentions)
Anti psd95 ab18258, by Abcam (1 mentions)
Fura 2 acetoxymethyl ester, by Thermo Fisher Scientific (1 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid, by Thermo Fisher Scientific (1 mentions)
45 protocols using 2 7 dichlorofluorescin diacetate dcfh da
1
Oxidative Stress Measurement in CSE-Exposed Cells
2
Amylin-induced calcium signaling in neurons
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Dulbecco’s modified Eagle’s medium, fetal bovine serum, fura-2-acetoxy-methyl ester, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glucose and PBS were obtained from Invitrogen (USA). Penicillin and streptomycin, NaCl, KCl, MgCl2, CaCl2 and glucose were purchased from Sigma (USA). CsA, JC-1, an In Situ BrdU-Red DNA Fragmentation (TUNEL) Assay Kit (ab66110), anti-TMEM119 (ab209064), anti-Iba1 (ab153696), anti-S100 (ab868), anti-MAP2 (ab11267) and anti-PSD95 (ab18258) were obtained from Abcam (USA). Donkey anti-mouse IgG (Cat#A-21202, Alexa Fluor 488), donkey anti-rabbit IgG (Cat#A-21206, Alexa Fluor 488) and donkey anti-rabbit IgG (Cat#A-21207, Alexa Fluor 594) were purchased from Thermo Fisher (USA). Finally, 2’,7’-dichlorofluorescin-diacetate (DCFH-DA) was purchased from Beyotime (China).
hAmylin (Tocris, UK) was dissolved to 500 μM in sterile water and immediately diluted with HEPES buffer (see calcium imaging methods) to a final concentration of 1 or 10 μM at room temperature. FAM-hAmylin was purchased from Shanghai Science Peptide Biological Technology Co., Ltd. (China). The sequence of hAmylin is KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (Modifications: Tyr-37 = C-terminal amide, Disulfide bridge between 2 - 7).
hAmylin (Tocris, UK) was dissolved to 500 μM in sterile water and immediately diluted with HEPES buffer (see calcium imaging methods) to a final concentration of 1 or 10 μM at room temperature. FAM-hAmylin was purchased from Shanghai Science Peptide Biological Technology Co., Ltd. (China). The sequence of hAmylin is KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (Modifications: Tyr-37 = C-terminal amide, Disulfide bridge between 2 - 7).
3
Multifunctional Nanomaterial-based Theranostics
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Chlorin E6, Atovaquone, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC HCl), and N-carboxythiosuccinimide (sulfo-NHS) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. Dimethyl sulfoxide (DMSO) was purchased from Beijing LABGIC technology Co., Ltd; monodisperse carboxylic magnetic mesoporous silica nanomaterials (Fe3O4@MSNs) were purchased from Nanjing NANOEAST Biotech Co., Ltd; targeted peptide (iRGD) was purchased from Hefei Bank Peptide Biological technology Co., Ltd; 1,3-diphenylisobenzofuran (DPBF) was purchased from Shanghai Macklin Biochemical Co., Ltd; PBS buffer solution was purchased from Beijing Rangeke Technology Co., Ltd; DMEM medium, peptide bovine serum (FBS), trypsin, EDTA-free trypsin and double antibody (PS) were purchased from Thermo Fisher Scientific. 4′,6-Diamidino-2-phenylindole (DAPI), 4% paraformaldehyde, and electron microscope fixative were purchased from Wuhan Servicebio Technology Co., Ltd; cck8 kit and AnnexinV-FITC/PI double staining apoptosis detection kit were purchased from Jiangsu KeyGEN BioTECH Co., Ltd; 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and calcein/PI cell activity and cytotoxicity detection kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd.
4
Anoikis Assay for Apoptosis
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DMEM medium and fetal bovine serum was obtained from Hyclone (Logan, UT). Cell Counting Kit-8 (CCK8) was from Dojindo (Kumamoto, Japan). Caspases activities detection kits, 2′,7′-dichlorofluorescin diacetate (DCFH-DA), and N-acetyl-L-cysteine (NAC) were purchased from Beyotime (Haimen, China). Z-VAD-FMK was from R&D Systems (Minneapolis, MN). Antibodies against protein tyrosine kinase 2/focal adhesion kinase (PTK2/FAK), p-PTK2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were the product of Cell Signaling Technology (Danvers, MA). Poly(2-hydroxyethyl methacrylate) (poly-HEMA) was produced by Sigma-Aldrich (St. Louis, MO). CytoSelect™ 24-Well Anoikis Assay kit was provided by Cell Biolabs (San Diego, CA). Caspase-3, 8 and 9 activity assay kits were provided by Beyotime Institute of Biotechnology (Haimen, China).
5
Intracellular ROS Measurement by DCFH-DA
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2′,7′-dichlorofluorescin diacetate (DCFH-DA) (Beyotime Biotechnology Co., Ltd., Shanghai, China) was used to assess the intracellular ROS with fluorescent microscopy. In brief, cells were pretreated with GSK205 10 μM for 1 h, and then stimulated with medium only or CSE for another 24 h. These cells then underwent incubation with 10 μM DCFH-DA for 20 min at 37°C. Finally, the intracellular ROS was assessed at 488/525 nm using fluorescent microscopy (Leica, Germany) and analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, MD, United States).
6
Intracellular ROS and 8-OHdG Quantification
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Intracellular ROS levels were evaluated using 2,7-dichlorofluorescin diacetate (DCFH-DA) (Beyotime Biotechnology). DCFH-DA can form the fluorescent compound, dichlorofluorescein, in the presence of ROS. First, cells were incubated with DCFH-DA (10 μM) for 20 min at 37° C; then, cells were washed at least five times with serum-free medium. Labeled cells were trypsinized, resuspended in PBS supplemented with 5% fetal bovine serum, and analyzed by flow cytometry (BD Biosciences). A minimum of 10,000 cells were analyzed per condition.
The level of 8-OHdG in cells was detected using an ELISA kit (Shanghai Yuanye Bio-Technology, Shanghai, China) as per the manufacturer's instructions. Concentrations were determined by comparing the optical density values to a standard curve.
The level of 8-OHdG in cells was detected using an ELISA kit (Shanghai Yuanye Bio-Technology, Shanghai, China) as per the manufacturer's instructions. Concentrations were determined by comparing the optical density values to a standard curve.
7
Synthesis and Characterization of Pyrene-Labeled Glutamate Conjugates
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H-Glu(OtBu)-OtBu·HCl and 1-hydroxybenzotriazole (HOBt) were purchased from J&K Chemical (China). 4-nitrophenyl chloroformate (NPC), triphosgene, cystamine dihydrochloride, γ-benzyl-L-glutamate, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC·HCl), 4-dimethylaminopyr idine (DMAP), and pyrene were purchased from Aladdin (Shanghai, China) and used as received. N,N’-dimethylformamide (DMF) and trimethylamine (TEA) were dried and filtered before use. Hoechst 33,342 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (USA). Bovine serum albumin, RPMI-1640 medium, and trypsin-EDTA were obtained from Hyclone (USA). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was obtained from Beyotime Institute of Biotechnology (Nantong, China). PTXL injections were supplied by Beijing Union Pharmaceutical Factory (Beijing, China). Alcohol, sodium methoxide, hydrochloric acid, and other chemicals were obtained from local commercial suppliers.
8
Oxidative Stress Assays in Lung Cells
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The content of malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by using MDA and SOD assay kits (Nanjing Jiancheng Bioengineering institute, Jiangsu, China) according to the manufacturer’s instruction. Intracellular reactive oxygen species (ROS) generation was measured by fluorescence dye 2′7’-dichlorofluorescin diacetate (DCFH-DA; Beyotime Institute of Biotechnology, China). Beas-2B and NHBE cells were plated into 12-well plates and pretreated for 2 h with 1, 5 and 10 μM ALT and then exposed with 5% CSE for 24 h. DCFH-DA solution (20 μM) was added to cells for 30 min and the fluorescence was measured by a fluorescence microscope (Invitrogen, WA, USA).
9
Rat L6 Skeletal Muscle Cell Analysis
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The rat L6 skeletal muscle cell was purchased from the Shanghai Institute of Life Sciences Cell Bank of Chinese Academy of Sciences; the Dulbecco's Modified Eagle's Medium and Fetal Bovine Serum from GIBCO, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) from the Beyotime Institute of Biotechnology China; 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-dglucose (2-NBDG) from Life Invitrogen USA. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) was purchased from Amresco USA.
10
Doxorubicin-Loaded Silica Nanoparticles
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The FeCl3·6H2O, L-glutamic acid, soybean phospholipids, polyvinylpyrrolidone (PVP) K-30, tetraethyl orthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), DOX, N-Hydroxysuccinimide (NHS), NH4Ac, ethylene glycol, chloroform, ethanol, and ammonium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was purchased from J&K (Atlanta, GA, USA). The RPMI-1640 culture medium and fetal calf serum (FCS) were obtained from Hyclone (Logan, UT, USA). The Hoechst 33342/propidium iodide (PI) double staining kit and 2,7-dichlorofluorescin diacetate (DCFH-DA) were provided by Beyotime Institute of Biotechnology (Jiangsu, China). The CellTiter-Glo® luminescent cell viability assay kit was purchased from Promega (Madison, WI, USA). All reagents were used directly as received without any further purification. Human esophagus carcinoma cells (Eca-109) were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China).
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