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DCFH-DA [2',7'-Dichlorodihydrofluorescein diacetate] *CAS 4091-99-0*

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Physical properties
Molecular weight487.29
Spectral properties
Excitation (nm)505
Emission (nm)526
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Excitation (nm)
Emission (nm)
DCFH-DA, 2',7'-Dichlorodihydrofluorescein diacetate (also called 2',7'-dichlorofluorescin diacetate), is hydrolyzed by cellular esterases to 2',7'-dichlorodihydrofluorescein (also called 2',7'-dichlorofluorescin) and is then oxidized to 2',7'-dichlorofluorescein primarily by H2O2. 2',7'-dichlorodihydrofluorescein diacetate might be reactive toward a broad range of oxidizing reactions that may be increased during intracellular oxidant stress. This probe is widely used to monitoring cellular redox processes.


Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
Recommended plateBlack wall/clear bottom
Instrument specification(s)FITC filter set

Example protocol


Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

2',7'-Dichlorodihydrofluorescein diacetate stock solution:
Make working solution in the concentration range of 1-10 mM in DMSO. Note: The unused DMSO stock solution should be aliquoted into a single use vial and stored at -20 °C. Keep from light.


2',7'-Dichlorodihydrofluorescein diacetate working solution:
Make working solution in the concentration range of 1-10 µM in a physiological buffer such as PBS, HBSS, HEPES buffers. Note: The optimal working concentration for your application must be empirically determined.


The following procedures provide a general guideline and should be modified for your particular application.

  1. Remove cells from growth media, add the dye working solution to the cells, and incubate the cells at room temperature or 37oC for 5 to 60 minutes.

  2. Remove the dye working solution; wash with pre-warmed HBSS, and add pre-warmed HBSS or growth medium and incubate at the optimal temperature. The optimal recovery time can vary widely, as some cell types normally exhibit low levels of esterase activity.

  3. Determine the baseline fluorescence intensity of a sample of the loaded cells prior to exposing the cells to experimental inducements. 6. Negative controls should be assessed as follows:

  4. Examine unstained cells for autofluorescence in the green emission range.

  5. For flow cytometry, ascertain that the forward and side scatter of cells is unchanged after dye-loading and treatment. Changes in cell dimensions may be related to blebbing or shrinkage resulting from handling or a toxic response.

  6. Examine the fluorescence of cell-free mixtures of dye and buffer/media with and without the inducer. In the absence of extracellular esterases and other oxidative enzymes, the gradual increase in fluorescence over time may be related to spontaneous hydrolysis, atmospheric oxidation, and/or light-induced oxidation.

  7. Examine the fluorescence of untreated (control) loaded cells that have been maintained in growth medium or simple buffer. In healthy cells, oxygen radicals are eliminated by cellular enzymes and/or natural antioxidants. Following the dye-loading recovery period, healthy cells should exhibit a low level of fluorescence that is relatively stable for the duration of the experiment; however, a gradual increase (due to auto-oxidation) or decrease (due to loss of dye from cells or photobleaching) in fluorescence may be observed. In the absence of any stimulus or inducement, a burst of fluorescence in healthy, untreated cells could indicate progress to cell death or some other oxidative event.

  8. Positive controls may be stimulated with H2O2 or tert-butyl hydroperoxide (TBHP) to a final concentration of ~100 µM (increase or decrease dose based on the sensitivity and response of the cells).


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of DCFH-DA [2',7'-Dichlorodihydrofluorescein diacetate] *CAS 4091-99-0* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM205.217 µL1.026 mL2.052 mL10.261 mL20.522 mL
5 mM41.043 µL205.217 µL410.433 µL2.052 mL4.104 mL
10 mM20.522 µL102.608 µL205.217 µL1.026 mL2.052 mL

Molarity calculator

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Spectral properties

Excitation (nm)505
Emission (nm)526



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