N-acetyl-L-cysteine, trolox, and 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-DCF-DA) were purchased from Sigma Aldrich and Invitrogen. Antibodies against cleaved caspase-3, -9, Poly ADP ribose polymerase (PARP), and anti-ZBTB3 were purchased from Cell Signaling Technology and Bethyl Laboratory. Anti-tubulin, PRDX2 and SOD2 antibodies were purchased from Millipore and Pierce.
Cm dcfda
Manufactured by Merck Group
Sourced in United States
CM-DCFDA is a fluorogenic dye that can be used to detect the presence of reactive oxygen species (ROS) in cells. It is a cell-permeant indicator for ROS that upon oxidation, emits fluorescence. The core function of CM-DCFDA is to serve as a tool for the measurement of intracellular ROS levels in various experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Mk571, by Merck Group (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (3 mentions)
Rhodamine 123, by Merck Group (3 mentions)
Ko143, by Merck Group (3 mentions)
Fl bopidy, by Merck Group (3 mentions)
Synergymx2, by Agilent Technologies (2 mentions)
Trolox, by Merck Group (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
Cyclosporine a, by Merck Group (1 mentions)
Facsverse, by BD (1 mentions)
Synergymx2 elisa plate reader, by Agilent Technologies (1 mentions)
5 protocols using cm dcfda
1
Oxidative Stress and Apoptosis Regulation
2
Quantifying Efflux Pump Activity in Cells
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Cells were incubated in the presence of 10 µM Rhodamine 123 (P-gp substrate, Sigma), FL-BOPIDY (BCRP substrate, Sigma), or CM-DCFDA (MRP substrate, Sigma) for 1 h at 37 ºC followed by cell lysis using RIPA buffer (ThermoFisher). For assessing the contribution of efflux pump in the drug uptake, cells were pre-incubated for 1 h in presence of 5 µM cyclosporine A (CsA, P-gp inhibitor, Sigma), 1 µM Ko143 (BCRP inhibitor, Sigma), or 10 µM MK571 (MRPs inhibitor, Sigma) and maintained during the incubation with drug efflux substrate. Following incubation, cells were briefly washed with ice-cold PBS and lysed with RIPA buffer. Fluorescence in cell lysates was assessed using a SynergyMX2 ELISA plate reader (Bio-Tek, Winooski, VT, USA). Relative fluorescence units (RFU) were normalized against the total protein content and the protein levels were determined by bicinchoninic acid assay (BCA, ThermoFisher). Fluorescence values (expressed as relative fluorescence unit or RFU) obtained from cell lysates in the absence of inhibitor (named as controls) were normalized to the protein content and expressed as RFU/µg protein.
3
Quantifying Intracellular Reactive Oxygen
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Both flow cytometric and spectrofluorometric techniques were used to detect intracellular ROS content. For 60 min, 5 μl of whole cell lysates were incubated at 37 °C with 5 mM dihydroethidium present in the media, following which, fluorescent signals were recorded at an excitation wavelength of 480 nm and an emission wavelength of 525 nm in a spectrofluorimeter (JASCO). A standard curve was prepared using increasing concentrations of dihydroethidium (DHE) and the intracellular ROS content were expressed as nanomoles of DHE produced/mg of protein. Intracellular ROS (H2O2) was estimated flow cytometrically using 5, 6-chloromethyl-2′7′ dichlorodihydrofluorescien diacetate (CM-DCFDA, Sigma Aldrich, USA). Tumor cells harvested from different experimental groups were incubated with DCFDA solution (25 μg/ml) for 30 min at 37 °C, washed and re-suspended in sterile PBS. ROS content was measured on a flow cytometer (BD FACS VERSE) using the emission intensity of 605 nm. A total of 10,000 events were acquired and analysis was done using FlowJo software (Version 10.7.2) [31 (link)].
4
Efflux Pump Inhibitor Assay
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Cells were incubated in the presence of 10µM Rhodamine 123 (P-gp substrate, Sigma), FL-BOPIDY (BCRP substrate, Sigma) or CM-DCFDA (MRP substrate, Sigma) for 1 hour at 37ºC followed by cell lysis using RIPA buffer (ThermoFisher). For assessing the contribution of e ux pump in the drug uptake, cells were pre-incubated for 1 hour in presence of 5µM cyclosporine A (CsA, P-gp inhibitor, Sigma), 1µM Ko143
(BCRP inhibitor, Sigma) or 10µM MK571 (MRPs inhibitor, Sigma) and maintained during the incubation with drug e ux substrate. Following incubation, cells were brie y washed with ice-cold PBS and lysed with RIPA buffer. Fluorescence in cell lysates was assessed using a SynergyMX 2 ELISA plate reader (Bio-Tek, Winooski, VT, USA). Relative uorescence units (RFU) were normalized against the total protein content and the protein levels were determined by bicinchoninic acid assay (BCA, ThermoFisher).
Fluorescence values (expressed as relative uorescence unit or RFU) obtained from cell lysates in the absence of inhibitor (named as controls) were normalized to the protein content and expressed as RFU/ µg protein.
(BCRP inhibitor, Sigma) or 10µM MK571 (MRPs inhibitor, Sigma) and maintained during the incubation with drug e ux substrate. Following incubation, cells were brie y washed with ice-cold PBS and lysed with RIPA buffer. Fluorescence in cell lysates was assessed using a SynergyMX 2 ELISA plate reader (Bio-Tek, Winooski, VT, USA). Relative uorescence units (RFU) were normalized against the total protein content and the protein levels were determined by bicinchoninic acid assay (BCA, ThermoFisher).
Fluorescence values (expressed as relative uorescence unit or RFU) obtained from cell lysates in the absence of inhibitor (named as controls) were normalized to the protein content and expressed as RFU/ µg protein.
5
Quantification of Efflux Pump Activities
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Cells were incubated in the presence of 10µM Rhodamine 123 (P-gp substrate, Sigma), FL-BOPIDY (BCRP substrate, Sigma) or CM-DCFDA (MRP substrate, Sigma) for 1 hour at 37ºC followed by cell lysis using RIPA buffer (ThermoFisher). For assessing the contribution of efflux pump in the drug uptake, cells were preincubated for 1 hour in presence of 5µM cyclosporine A (CsA, P-gp inhibitor, Sigma), 1µM Ko143 (BCRP inhibitor, Sigma) or 10µM MK571 (MRPs inhibitor, Sigma) and maintained during the incubation with drug efflux substrate. Following incubation, cells were briefly washed with ice-cold PBS and lysed with RIPA buffer. Fluorescence in cell lysates was assessed using a SynergyMX 2 ELISA plate reader (Bio-Tek, Winooski, VT, USA). Relative fluorescence units (RFU) were normalized against the total protein content and the protein levels were determined by bicinchoninic acid assay (BCA, ThermoFisher). Fluorescence values (expressed as relative fluorescence unit or RFU) obtained from cell lysates in the absence of inhibitor (named as controls) were normalized to the protein content and expressed as RFU/µg protein.
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