HK2 cells were cultured in 24-well plates until they reached confluence. Cells were pre-treated with TAPI1 (1 µM for 30 min pre-trement, Calbiochem) and TAPI2 (10 µM for 1hr pretreatment, Cayman) and then treated with 10 µg/ml of LPS for 8 h. At the end of the experimental periods, cells were preloaded with 10 µM 2′, 7′-dichlorofluorescein diacetate (DCF-DA; Molecular Probes) for 30 min at 37℃. Fluorescence intensity was analyzed by a fluorescence reader (Fluoroscan Ascent FL; Lab systems, Helsinki, Finland) using 485 nm excitation and 538 nm emission filter. HK2 cells were cultured on a 6-well plate for DCF-DA staining. Cells were pre-treated with TAPI1 (1 µM for 30 min) and TAPI2 (10 µM for 1 h) and then treated with 10 µg/ml of LPS for 8 h. Cells were washed twice with hanks balanced salt solution (HBSS) and incubated with HBSS (without phenol red) containing DCF-DA for 30 min at 37℃ in dark. The images were obtained with a fluorescence microscope (Nikon, Tokyo, Japan).
2 7 dichlorofluorescein diacetate dcf da
Manufactured by Thermo Fisher Scientific
Sourced in United States
2′,7′-dichlorofluorescein diacetate (DCF-DA) is a fluorogenic compound used to measure the level of reactive oxygen species (ROS) in cells. It is a cell-permeant indicator that is non-fluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell.
Automatically generated - may contain errors
Lab products found in correlation
Cumene hydroperoxide, by Merck Group (5 mentions)
Dodecyl maltoside, by Merck Group (5 mentions)
Coenzyme q2, by Merck Group (5 mentions)
Antimycin a, by Merck Group (5 mentions)
Cytochrome c, by Merck Group (5 mentions)
Glutathione reductase, by Merck Group (4 mentions)
Nadph, by Merck Group (4 mentions)
Erythromycin, by Merck Group (4 mentions)
N acetyl l cysteine, by Merck Group (4 mentions)
Aspirin, by Merck Group (3 mentions)
1 chloro 2 4 dinitrobenzene, by Merck Group (3 mentions)
Atp bioluminescent somatic cell assay kit, by Merck Group (3 mentions)
Apoptosis detection kits for flow cytometry, by BD (3 mentions)
Resorufin, by Merck Group (3 mentions)
7 ethoxyresorufin, by Merck Group (3 mentions)
Mitosox red, by Thermo Fisher Scientific (3 mentions)
U0126, by Cell Signaling Technology (3 mentions)
N acetyl cysteine (nac), by Merck Group (3 mentions)
5 5 dithiobis 2 nitrobenzoic acid, by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
40 protocols using 2 7 dichlorofluorescein diacetate dcf da
1
LPS-Induced Oxidative Stress Assay
2
Synthesis and Characterization of Antimicrobial Peptides
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FNR-675 N-hydroxysuccinimide (NHS) ester was purchased from BioActs (Incheon, Korea). SuperdexTM 200 (10/300); CM sepharose® Fast Flow and DEAE sepharose® Fast Flow were bought from GE Healthcare (Piscataway, NJ, USA). The 2′,7′-dichlorofluorescein diacetate (DCF-DA) and MitoSOX Red were obtained from Molecular Probes Inc., (Eugene, OR, USA). Histatin 5 and melittin were synthesized by using a microwave peptide synthesizer (Discover BioTM, CEM Co., Matthews, NC, USA) via solid phase method. All other chemicals were of reagent grade and all solvents were HPLC grade.
3
Quantifying Intracellular Oxidative Stress in C. elegans
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For measuring intracellular ROS levels, synchronized YA worms were collected and washed thrice in 1X M9 buffer. Worm extracts were prepared by flash freezing worm pellets in liquid nitrogen and freeze-thawing for three cycles, followed by sonication (setting of 30 amplitudes, 12 cycles of 1 s pulse on/off, 5–8 times using Misonix Ultrasonic processor 4000) in 1X PBS. The worm extract was centrifuged at 20,000 × g for 15 min at 4 °C, and the protein concentration of the supernatant was determined using Bradford protein estimation kit (Bio-Rad, USA). Supernatant containing 5 μg of protein was pre-incubated with 50 μM of 2,7-dichlorofluorescein diacetate (DCFDA, Molecular Probes, USA) in 100 μL of 1X PBS at 37 °C for 1 h. Fluorescence intensity was measured in FLUOstar Omega (BMG Labtech, USA) every 10 min for 1 h at excitation wavelength 485 nm and emission wavelength 520 nm.
4
Quantitative ROS Detection Assays
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(a) The total intracellular superoxide and hydroxyl radical in cells was detected using Cellular Reactive Oxygen Species Detection Assay Kit (ab186027, Abcam, Cambridge, UK), following the manufacturer’s instructions. Briefly, cells were seeded into 96-well plate with 2 × 104 cells per well. Add 100 µL/well of ROS Red Working Solution into the plate. Incubate cell plate in a 37 ℃/5% CO2 incubator for 1 h, and then detect the fluorescence signal by microplate reader.
(b) Intracellular hydrogen peroxide detection. Cells were plated into 12-well plates with different treatments. Each well was loaded with 30 μm of the H2O2-sensitive fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCF-DA; Molecular Probes, Leiden, The Netherlands) and then the plate was plated at 37 ℃ for 4 h. The cells were washed with PBS for three times and then the intracellular fluorescence was quantified using flow cytometry.
(c) The level of superoxide anion was determined by ethidium fluorescence. Briefly, cells were seeded into 12-well plates wit different treatments. The cells were incubated with 1 mM hydroethidine at 37 ℃ for 5 min and then washed three times by PBS. The cells were then resuspended and subjected to flow cytometry analysis.
5
Lipid-Induced Fluorescent Probes Quantification
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SYTOX-Green, 2′,7′-dichlorofluorescein diacetate (DCF-DA) and MitoSOX Red were purchased from Molecular Probes Inc., (Eugene, OR, USA). Phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Ergosterol (Erg), cholesterol (CH), and sphingomyelin (SM) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). FAM N-hydroxysuccinimide (NHS) ester was purchased from BioActs (Incheon, Korea). All other reagents were of analytical grade.
6
Evaluating Oxalate and C-Phycocyanin Effects
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Oxalate, C-phycocyanin (CP), DMEM (Dulbecco's Modified Eagle Medium), fetal bovine serum (FBS), propidium iodide (PI), trypan blue, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), dimethyl sulfoxide, trypsin, Tween 20, and Triton X-100 were purchased from Sigma Chemicals (St. Louis, MO, USA). 2, 7–dichlorofluorescein diacetate (DCF-DA) (Molecular probes, Inc. USA), and JC-1 dye (Molecular probes, Inc. USA) were procured from Molecular Probes, Inc. (USA), P-JNK/SAPK and P- ERK1/2 antibodies from Cell signaling Technology, USA. T-JNK and T-ERK1/2 antibodies were purchased from Santa Cruz Biotechnology, INC, USA.
7
Natural Product Library Cytotoxicity Screening
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The natural product library was provided by Korea Institute of Science and Technology (KIST; Gangneung, Korea). The extract library was composed of ethanolic extracts of Korean native plants. MTT (tetrazolium bromide), dimethyl sulfoxide (DMSO), and N-Acetyl-L-Cysteine (NAC) were purchased from Sigma (St. Louis, MO). 2′,7′-Dichlorofluorescein diacetate (DCF-DA) was purchased from Molecular Probes (Eugene, OR). The TNF-α was purchased from Peprotech (Rocky Hill, NJ). The anti-PARP-1, anti-ATF4, anti-caspase-7, anti-actin, and anti-GAPDH antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-CHOP, anti-phospho-NF-κB, anti-NF-κB, anti-IL-1β, anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho-eIF2α, anti-eIF2α, and horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA). Annexin V-flamma 488 was purchased from Bioacts (Korea). Hoechst 33342 was purchased from Invitrogen (CA).
8
Comparative Analysis of Lung Cancer Cell Cytokine
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All cell lines were sourced from the American Type Culture Collection (ATCC, Manassas, VA). These included seven lung cancer cells, H1437, H522, H1650, A549, H460, H1299 and 196, and one normal human lung fibroblast cell line, IMR-90. c. For the human lung fibroblast IMR-90 cells ranging from 29 to 34 in population doubling level (PDL) were used. Cell lines were maintained in Eagle's minimal essential media (EMEM, Gibco-Invitrogen, Grand Island, NY) and contained 10 % (15 % for IMR-90 cells) heat-inactivated FBS (Gibco-Invitrogen), sodium bicarbonate (2 mg/ml; Sigma-Aldrich, St Louis, MO), penicillin (100 units/ml), and streptomycin (100 μg/ml; Gibco-Invitrogen). An ELISA kit (R&D Systems) was used to assay IL-6 production, according to the manufacturer's instructions. The concentrations of IL-6 were normalized to the cell numbers in the cell culture. 2′,7′- dichlorofluorescein diacetate (DCF-DA) was obtained from Molecular Probes. MK 2206 was obtained from selleckchem. N-acetylcystein (NAC) was obtained from Gibco-Invitrogen.
9
Investigating Oxidative Stress Mechanisms
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Unless otherwise specified, reagents were purchased from Sigma-Aldrich (St. Louis, MO). Ferritin heavy chain (FTH1) antibody RabMAb, HO-1 antibody and catalase antibody were purchased from Abcam (Cambridge, MA). Toll like receptor 4 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Protease Inhibitor Cocktail Set V, EDTA-free was from Calbiochem (Darmstadt, Germany). HO-1 siRNA (ID#194530), catalase siRNA (ID#2444) and toll like receptor 4 siRNA (ID#14195) were purchased from Applied Biosystems (Carlsbad, CA). Lipofectamine RNAimax was purchased from Invitrogen (Grand Island, NY). Necrostatin-1 was purchased from Selleckchem (Houston, TX). Alamar blue was from AbD Serotec (Raleigh, NC). 2’,7’–Dichlorofluorescein diacetate (DCFDA) was purchased from Molecular Probes (Carlsbad, CA).
10
Inflammatory Pathways Modulation Protocol
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Lipopolysaccharide (LPS), Chenodeoxycholic acid (CDCA), CA-074 Me, Tyrphostin AG 1478, A438079 and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO). SQ22536 was obtained from Abcam (Cambridge, MA). Cytochalasin D and Z-Guggulsterone were purchased from Santa cruz Biotechnology (Santa cruz, CA). VX-765 (belnacasan) and MK-2206 were purchased from Selleck ( Houston, TX). SP600125 and U0126 were obtained from Cell Signaling Technologies (Beverly, MA). RPMI 1640, DMEM and antibiotics were obtained from Invitrogen (Carlsbad, CA). 2’, 7’-dichlorofluorescein diacetate (DCF-DA) were from Invitrogen/Molecular Probes. ELISA Kits were purchased from eBioscience (San Diego, CA).
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