Cell culture supplies including DMEM/F-12 media, RPMI 1640 media, penicillin/streptomycin, L-glutamine, fetal bovine serum (FBS), CM-H2DCFDA dye and MitoSox® dye were purchased from Invitrogen. Rotenone, mouse anti-β actin antibody, mitoTEMPO, disuccinimidyl suberate (DSS), BSA lyophilized powder and acridine orange (AO) were purchased from Sigma-Aldrich. Lipopolysaccharide (E. Coli O111:B4) (LPS) and c-Abl antibody were purchased from EMD Millipore. Dasatinib (DAS), p-c-Abl (pY412), PKCδ, p-PKCδ (pY311), phospho-IκBα, iNOS, ASC, TOM20, lamin B, tubulin and p65 antibodies were purchased from Santa Cruz Biotechnology. Beclin1, NLRP3, caspase-1, LC3B, p-c-Abl (pY245), IL-1β and IL-18 antibodies were purchased from Cell Signaling Technology. TFEB antibody was Bethyl laboratories, Inc. Caspase 1 (p10) antibody was purchased from Adipogen. The mouse IL-1β and IL-18 ELISA kits from eBiosciences.
Cm h2dcfda dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
CM-H2DCFDA dye is a fluorescent indicator used to detect the presence of reactive oxygen species (ROS) in biological samples. It is a cell-permeant indicator that is non-fluorescent until it is oxidized by ROS, at which point it emits a fluorescent signal. This dye can be used to monitor oxidative stress in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Facs lsrii flow cytometer, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Synergy h1 hybrid multi mode microplate reader, by Agilent Technologies (2 mentions)
Acridine orange, by Merck Group (1 mentions)
Mitosox dye, by Thermo Fisher Scientific (1 mentions)
Disuccinimidyl suberate dss, by Merck Group (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Tom20, by Santa Cruz Biotechnology (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
Phospho iκbα, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β actin antibody, by Merck Group (1 mentions)
Bsa lyophilized powder, by Merck Group (1 mentions)
Mitotempo, by Merck Group (1 mentions)
Caspase 1, by Cell Signaling Technology (1 mentions)
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Lamin b, by Santa Cruz Biotechnology (1 mentions)
Nlrp3, by Cell Signaling Technology (1 mentions)
35 protocols using cm h2dcfda dye
1
Oxidative Stress Signaling Pathways
2
Eugenol-Induced ROS Measurement
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK-293 cells or NIH-3T3 cells were seeded into 6-well plates at a density of 3x105 cells/well in fully supplemented DMEM for 24 h before they were treated with 50 µg/ml eugenol for 6 h at 37˚C. Untreated and eugenol-treated cells were subsequently harvested in fully supplemented DMEM, centrifuged for 5 min at 200 x g and 4˚C and suspended in Dulbecco's PBS (DPBS) containing the 10 µM CM-H2DCFDA dye (Invitrogen; Thermo Fisher Scientific, Inc.) with or without 50 µM H2O2, at 37˚C in the dark for 30 min. The cells were then pelleted and resuspended in ice-cold DPBS prior to the addition of propidium iodide (1 µg/ml) to the cells for 5 min at room temperature, for analysis by flow cytometry (MoFlo® flow cytometer; Beckman Coulter, Inc.) by measuring fluorescence emission at 525 nm (PI) following excitation at 488 nm (DCFDA/FITC). Since live cells are impermeant to propidium iodide, only cells negative for propidium iodide staining were assessed for ROS production. Data was analyzed by Kaluza C Analysis software 2.1 (Beckman Coulter, Inc.).
3
Quantifying Intracellular Reactive Oxygen Species
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The level of ROS was measured using the CM-H2DCFDA dye (Invitrogen, Carlsbad, CA, USA). CM-H2DCFDA was dissolved in dimethylsulfoxide to obtain a 10 mM dye, which was then diluted in PBS containing Ca2+ and Mg2+ to achieve the final concentration of 10 μM. Cells were seeded in a 96-well black plate at a density of 5 × 104 cells/well. After overnight incubation, cells were pretreated with various concentrations of the PCS extract and subsequently treated with MGO (1 mM) for 1 h. The cells were then fixed in 10% neutral-buffered formalin for 10 min at room temperature, followed by staining with 10 μM CM-H2DCFDA for 30 min at 37°C. After staining, cells were washed twice with PBS containing Ca2+ and Mg2+, and the plate was covered with aluminum foil. Fluorescent intensity was measured immediately at an excitation/emission wavelength of 495/527 nm using a fluorescence microplate reader.
4
Quantifying Oxidative Stress in 661w Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A general oxidative stress indicator, CM-H2DCFDA, was used to examine the change in ROS levels in the 661w cell line. After 48 h incubation, the 661w cells were harvested. The cells pallets were resuspended in 300 μL PBS and divided into two equal portions. One portion of the cells was stained with 50 μL CM-H2DCFDA dye (C6827, Invitrogen) at a working concentration of 10μM, and the second was stained with Hoechst 33342, a nuclear dye, for normalization. The cells were then incubated at 37 °C for one hour and protected from light. Subsequently, the cells were washed with PBS, centrifuged at 210× g for 6 min, and lysed with buffer containing 0.75 M HCL and 0.2% Triton-X 100 at room temperature for 30 min in darkness. The supernatants were collected after centrifuging the cell lysate at 21,380× g for 5 min, and 100 μL was added to a well of a 96-well plate with a technical replicate. The fluorescence level was measured by a CLARIOstar® microplate reader (BMG LABTECH, Ortenberg, Germany).
5
ROS Detection in Cell Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Approximately 50,000 cells in complete cell culture medium (CTCM) were seeded per well in a 96-well plate. After infection, cells were incubated with 10 μM CM-H2DCFDA dye (Invitrogen) in the dark at 37°C for 30 mins. Cells were washed with Hank's balanced salt solution (HBSS) with calcium and magnesium. Fluorescence was measured using a Fluostar Omega (BMG Biotech) fluorimeter with setup excitation at 485 nm and emission at 540 nm.
6
Evaluating Cellular ROS Production by Fluorescence Microscopy
7
Evaluating Cellular Responses to Bioactive Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dimethyl sulfoxide (DMSO), trypan blue, paraformaldehyde, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), ganoderic acid A, ganoderic acid G, lucidenic acid A, and ergosterol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's Modified Eagle Medium, fetal bovine serum, 4′,6-diamidino-2-phenylindole dye (DAPI), dihydroethidium (DHE), fluorescein phalloidin dye, annexin-V-conjugated fluorescein isothiocyanate (FITC) dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1) dye, propidium iodide (PI) dye, bicinchoninic acid assay (BCA) kit, phenylmethylsulphonyl fluoride, CM-H2DCFDA dye, MitoSOX Red, penicillin/streptomycin, protease inhibitor cocktail, 0.25% (w/v) trypsin containing 1 mM EDTA, and phosphate-buffered saline (PBS) were all purchased from Invitrogen (Carlsbad, CA, USA). Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) and lactate dehydrogenase (LDH) assay kits were obtained from Roche (Basel, Switzerland). Primary antibodies against Nrf2 and Keap1 were obtained from Abcam (Cambridge, UK). Other primary antibodies and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
8
Gingerol-induced ROS Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gingerol induced ROS generation was evaluated by staining cells with CM-H2-DCFDA dye (Invitrogen, Carlsbad, CA, USA). Cells were harvested by trypsinization, pretreated with or without NAC (4 mM) at 37°C for 1 h and washed with PBS twice. Thereafter cells were resuspended in serum free media and stained with 5 mM CM-H2-DCFDA in dark with continuous shaking for 30 min. Stained cells were washed and resuspended in serum free media. Both NAC pretreated and untreated cells were treated with 6-gingerol and fluorescence was measured in slow kinetics mode for 6 h in BMG Fluostar Omega spectrofluorimeter.
9
Evaluating Cell Stress Responses
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CM-H2DCFDA dye, Fluo-4 AM calcium dye (Molecular Probes, United States), and JC-1 dye (Molecular Probes, United States) were employed to assess ROS, calcium, and MMP, respectively. The fluorescent images of calcium and JC-1 were captured by a confocal microscope. H9c2 cells (1 × 104/well) were seeded in μ-Slide 8-well glass bottom plates. Cells were labeled by Fluo-4 AM or JC-1 probe for 30 min after LS102 treatment according to the manufacturer’s protocols. The fluorescent intensity of images was analyzed by using the Image J software and a randomly captured method was used to make sure there were three fields per group. The fluorescence intensity of calcium, ROS were detected by the flow cytometer. All cells in the 6-well plates were collected and then suspended in a pre-warmed DMEM buffer containing CM-H2DCFDA or Fluo-4 AM calcium dye. Each group (1 × 104 cells) intensity was calculated as representation.
10
ROS Detection in Skeletal Muscle
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For ROS detection, fresh-frozen TA muscle cryosections (8 μm) were incubated for 30 min at 37°C with 10 μM CM-H2DCF-DA dye (Molecular Probes, Eugene, Oregon, USA) in Hank's buffered salt solution. Then, the muscles were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. The nuclei were stained with Hoechst 33258 (1 : 5,000) in PBS. After rinsing, the muscles were mounted with a fluorescent mounting medium (Dako, USA) under a glass slide and viewed and photographed using the Motic BA310 epifluorescence microscope [31 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!