活性氧(ROS)检测试剂盒(探针法)

Reactive Oxygen Species (ROS) Detection Kit (Probe assay)

Molecular Probes offers derivatives of reduced fluorescein and calcein as cell-permeant indicators for reactive oxygen species. Chemically reduced and acetylated forms of 2′,7′-dichlorofluorescein (DCF) and calcein are nonfluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell. Esterase cleavage of the lipophilic blocking groups yields a charged form of the dye that is much better retained by cells than is the parent compound. Oxidation of these probes can be detected by monitoring the increase in fluorescence with a flow cytometer, fluorometer, microplate reader, or fluorescence microscope, using excitation sources and filters appropriate for fluorescein (FITC). Because the dyes are susceptible to photo-oxidation, low light conditions should be used for fluorescence microscopy applications whenever possible. 

The carboxy derivative of fluorescein, carboxy-H2DCFDA, carries additional negative charges that improve its retention compared to noncarboxylated forms. The fluorinated derivative, H2DFFDA, exhibits improved photostability compared to chlorinated fluorescein derivatives. Derivatives with a thiol-reactive chloromethyl group or an amine-reactive succinimidyl ester group allow for covalent binding to intracellular components, permitting even longer retention within the cell. H2DCFDA, SE may also provide extracellular labeling. 

Dihydrocalcein, AM is freely permeant to cell membranes and is oxidized to green-fluorescent calcein, which has excellent retention properties. However, unlike the fluorescein derivatives, calcein fluorescence can be quenched by Fe3+, Co2+, Ni2+, and Cu2+ at pH 7. Other reduced fluorescein ROS indicators available from Molecular Probes include aminophenyl fluorescein and hydroxyphenyl fluorescein.

Simplified Protocols

1.1 Wash cells with 1×PBS (RT) two times;

1.2 Add 100μl 1×PBS (RT) containing 50 μM probe and lay for 30 minutes in cell incubator;

1.3 Remove probe by wash cells two times with 1×PBS (RT)，add 100μl culture medium with fetal bovine serum and lay for 10 minutes in cell incubator;

1.4 Observe fluorescence by microscope.

Detailed Protocols

The following protocols provide general guidelines derived from various publications, and should be modified for the particular application and sensitivity required.

1.1 Shortly before performing the experiments, reconstitute the ROS indicator to make a concentrated stock solution. Keep tightly sealed until ready to use.

1.2 Remove cells from growth media via centrifugation or pipetting. Resuspend cells in prewarmed buffer (PBS, HBSS, HEPES, or other simple physiological buffer) containing the probe to provide a final working concentration of ~1–10 mM dye. The optimal working concentration for your application must be empirically determined.

1.3 Incubate at the optimal temperature for the cells. Generally, a loading time of 5–60 minutes is sufficient.

1.4 Remove the loading buffer; return the cells to prewarmed growth medium and incubate at the optimal temperature. For derivatives with acetoxymethyl ester (AM) and/or diacetate groups, allow a short recovery time for cellular esterases to hydrolyze the AM or acetate groups and render the dye responsive to oxidation. The optimal recovery time can vary widely, as some cell types normally exhibit low levels of esterase activity.

1.5 Determine the baseline fluorescence intensity of a sample of the loaded cells prior to exposing the cells to experimental inducements. If using the succinimidyl ester derivative, any extracellularly bound dye can be quenched using Trypan Blue (~0.0025% w/v) in order to better distinguish the signal from the intracellular ROS response.

1.6 Negative controls should be assessed as follows:

a) Examine unstained cells for autofluorescence in the green emission range.

b) 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.

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

d) Examine the fluorescence of untreated 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;

Reactive Oxygen Species (ROS) Detection Reagents

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.

1.7 To create positive controls, oxidative activity may be stimulated with:

a) the tumor promoter 4b-phorbol 12-myristate 13-acetate (PMA; stock solution 1 mM in DMF; working concentration 100 pM to 10 mM)

b) the bacterial chemotatic peptide N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP; stock solution 1 mM in DMF; working concentration 1–10 mM)

c) H2O2 or tert-butyl hydroperoxide (TBHP) to a final concentration of ~100 mM (increase or decrease based on the sensitivity and response of the cells).

1.8 To assure that an added drug or compound will not quench the dye, examine the absorbance spectrum of the compound and determine that the absorbance peak does not overlap with either the excitation or emission peak of the oxidized dye. Alternatively, you can mix a solution of the drug/compound with carboxy-DCF (C368), fluorescein (F1300), or calcein (C481), then compare the fluorescence intensity to a control solution of the dye, or subject a culture loaded with the cell-permeant, oxidized form of the dye to the drug/compound and compare to dye-loaded cells untreated.

注意事项

1. The probe (component A) is air sensitive and should be stored under dry argon or nitrogen. Both probe and control should be protected from light.

2. Avoid freeze-thaw cycles. This product is stable for at least 3 months.