How does CM H2DCFDA work?
CM-H2DCFDA passively diffuses into cells, where its acetate groups are cleaved by intracellular esterases and its thiol-reactive chloromethyl group reacts with intracellular glutathione and other thiols.
What is H2DCFDA?
The cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (also known as dichlorofluores cin diacetate) is a chemically reduced form of fluorescein used as an indicator for reactive oxygen species (ROS) in cells, for example to detect the generation of reactive oxygen intermediates in neutrophils and …
What is the principle of DCFH-DA assay?
The DCFDA assay protocol is based on the diffusion of DCFDA / H2DCFDA / DCFH-DA / DCFH into the cell. It is then deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2′, 7′ –dichlorofluorescein (DCF).
What is DCF fluorescence?
DCF is often used as a fluorescent indicator of ROS formation in both simple biochemical antioxidant assays and in cell culture based antioxidant assays. DCF can be oxidized by peroxynitrite, hydroxyl radicals, and peroxidase to generate a highly fluorescent end product.
How do you test for ROS?
Assess ROS by immediately analyzing your cells by flow cytometry using the FL1 channel (green fluorescence), or by fluorescence plate reader, or by fluorescence microscopy. The fluorescence can be detected by using excitation and emission wavelengths that are appropriate for green fluorescence.
How do you dissolve H2DCFDA?
It can be dissolved in EtOH, DMSO and DMF at least 10 mg/ml. DMF is probably a better solvent for better stability. The stock solution is stable at least 6 months at –20°C. If H2DCFDA is prepared in aqueous buffer, dissolve before in 0.1M Na2CO3 and then in PBS to the desired concentration or pH.
What is DCF in biology?
Dichlorofluorescein (DCF) is an organic dye of the fluorescein family, being substituted at the 2 and 7 positions by chloride. It is used as an indicator for argentometry by Fajans method.
Where is flow cytometry performed?
Unless your healthcare provider gives you specific instructions, there is usually no need for preparation before having a flow cytometry analysis. You will simply visit your provider’s office where they will obtain a blood, bone marrow or tissue sample.
What is the cm-h2dcfda protocol?
Detection of Intracellular Reactive Oxygen Species (CM-H2DCFDA) 1 Authors 2 Summary. This protocol describes a method to detect intracellular ROS level in living cells. 3 Introduction. Reactive oxygen species (ROS) play a critical role in cellular physiopathology. 4 Materials 5 Procedure. 6 Anticipated results. 7 References.
What is H2 DCFDA used for?
Description The cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H 2 DCFDA) (also known as dichlorofluores cin diacetate) is a chemically reduced form of fluorescein used as an indicator for reactive oxygen species (ROS) in cells, for example to detect the generation of reactive oxygen intermediates in neutrophils and macrophages.
How is h2dcfda converted to DCF after cleavage?
The cell-permeant H2DCFDA passively diffuses into cells andis retained in the intracellular level after cleavage by intracellular esterases. Upon oxidation by ROS, th e nonfluorescent H2DCFDA is converted to the highly fluorescent 2′, 7′-dichlorofluorescein (DCF).
What is the full form of h2dcfda?
The cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (also known as dichlorofluores cin diacetate) is a chemically reduced form of fluorescein used as an indicator for reactive oxygen species (ROS) in cells, for example to detect the generation of reactive oxygen intermediates in n