What is CM H2DCFDA?
Description. CM-H2DCFDA is chloromethyl derivative of H2DCFDA useful as indicator for reactive oxygen species (ROS) in cells.
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 does DCFDA measure?
DCFDA / H2DCFDA / DCFH-DA / DCFH is a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell.
What is Ros assay?
The OxiSelect™ ROS Assay Kit is a cell-based assay for measuring hydroxyl, peroxyl, and other reactive oxygen species activity within a cell. The assay employs the cell-permeable fluorogenic probe DCFH-DA, which diffuses into cells and is deacetylcated by cellular esterases into the non-fluorescent DCFH (Figure 1).
What does reactive oxygen species do?
A type of unstable molecule that contains oxygen and that easily reacts with other molecules in a cell. A build up of reactive oxygen species in cells may cause damage to DNA, RNA, and proteins, and may cause cell death. Reactive oxygen species are free radicals.
What color is propidium iodide?
red-fluorescent
Propidium iodide (PI) is a popular red-fluorescent nuclear and chromosome counterstain.
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.
What is the principle of DCFH DA assay?
Mechanistically, DCFH-DA is taken up by cells where cellular esterase cleaves off the acetyl groups, resulting in DCFH. Oxidation of DCFH by ROS converts the molecule to DCF, which emits green fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.
What are ROS levels?
Oxidative stress refers to elevated intracellular levels of reactive oxygen species (ROS) that cause damage to lipids, proteins and DNA. Oxidative stress has been linked to a myriad of pathologies. However, elevated ROS are also signaling molecules i.e. redox biology that maintain physiological functions.
How do you measure ROS?
The intracellular levels of ROS can be measured by flow cytometry using dihydrofluorescein diacetate (DCFH) to detect intracellular hydrogen peroxide radicals. This dye is oxidized to the highly fluorescent derivative dichlorofluorescein (DCF), which is detected by the use of flow cytometer [46,46,48, 82].
Are reactive oxygen species good or bad?
Reactive oxygen species (ROS) can cause damage to the basic building blocks of the cell including DNA, protein and lipids. (A) DNA damage can occur in the form of double stranded breaks as a result of ROS-induced conversion of guanine to 8-oxoguanine.
How do you dissolve propidium iodide?
Propidium iodide (PI) is supplied as a crystalline solid. A stock solution may be made by dissolving the PI in the solvent of choice. PI is soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide, which should be purged with an inert gas.
What is the advantage of chloromethyl derivative of h2dcfda?
The chloromethyl derivative of H2DCFDA (CM-H2DCFDA) provides much better retention in live cells than H2DCFDA. Dead or dying cells produces ROS.
What is the difference between h2dcfda and cm-h2 DCFDA?
CM-H2DCFDA is a chloromethyl derivative of H2DCFDA, useful as an indicator for reactive oxygen species (ROS) in cells. This indicator exhibits much better retention in live cells than H2DCFDA. CM-H2DCFDA passively diffuses into cells, where its acetate
How do you convert h2dcfda to fluorescent H2D?
H 2 DCFDA passively diffuses into the cell, and upon oxidation by ROS, the non-fluorescent H 2 DCFDA is converted to fluorescent 2 ′ ,7 ′ -dichlorofluorescein [22].
Why is my h2dcfda dye not working?
Some dyes, such as H2DCFDA, require esterase cleavage, so don’t incubate in the presence of serum (which contains esterases that can prematurely cleave the dye). If your positive control does not show significant change compared to the negative control, try increasing the concentration and label time for the dye.