Reactive Oxygen Species Assay Kit

Cat. No.: R272916

Overview

Product composition

	Product name	Package
Liquid A	H2DCFH-DA(10mM)	0.1mL
Liquid B	Active oxygen positive control(Rosup,50mg/mL)	1mL

Product Introduction

Reactive Oxygen Species Assay Kit (Reactive Oxygen Species Assay Kit) is a kit that uses fluorescent probe H2DCFH-DA for reactive oxygen detection. H2DCFH-DA itself has no fluorescence and can freely pass through the cell membrane. After entering the cell, it can be hydrolyzed by intracellular esterase to produce DCFH. DCFH cannot penetrate the cell membrane, so that the probe is easily loaded into the cell. The reactive oxygen species in cells can oxidize non-fluorescent DCFH to produce fluorescent DCF. Detecting the fluorescence of DCF can know the level of reactive oxygen species in the cell. According to the fluorescence produced in living cells, the content and changes of reactive oxygen species can be judged. It can be directly observed with a flow cytometer or a fluorescence microscope. It is a classic method for detecting reactive oxygen species in tissues or living cells. This kit provides Rosup, a positive control reagent for active oxygen, to facilitate the detection of active oxygen. Rosup is a mixture with a concentration of 50 mg/mL. Rosup is a reactive oxygen-inducing drug. According to its fluorescence signal intensity, the true level of reactive oxygen can be analyzed.

The kit has low background, high sensitivity, wide linear range and convenient use. This kit can measure 1000 samples 1000T (96 well plate).

Precautions

1. After the probe is loaded, be sure to wash the remaining probes that have not entered the cell, otherwise it will cause a high background.

2. The positive control Rosup generally uses a concentration of 100 μM (the recommended concentration is 100-400 μM, depending on the cell type). Usually, a significant increase in reactive oxygen species can be observed 0.5-4h after stimulation. For different cells, the effect of reactive oxygen species positive control may be quite different. If no increase in active oxygen is observed within 30 minutes after stimulation, the induction time can be extended or the concentration of active oxygen positive control can be appropriately increased. If the reactive oxygen species increase too quickly, shorten the induction time or appropriately reduce the concentration of the reactive oxygen species positive control.

3. For some special cells, if the negative control cells without stimulation are found to have stronger fluorescence during the experiment, you can dilute DCFH-DA at 1:2000-1:5000 to make the final concentration of DCFH-DA 2-5 μM. The loading time of the probe can also be appropriately adjusted within 15-60 min according to the situation.

4. Active oxygen positive control (Rosup) is only used as a positive control sample, and it is not necessary to add active oxygen positive control to every sample.

5. After the probe is loaded and the remaining probes are cleaned, scan the excitation wavelength and emission wavelength to confirm whether the probe is well loaded. Please refer to the figure above for the excitation and emission spectra of DCF.

6. Try to shorten the time from probe loading to measurement (except stimulation time) to reduce all possible errors.

7. For your safety and health, please wear lab coats and disposable gloves.

8. If it is quantitative, make a standard curve. First make a different concentration of H2O2 oxidized DCFA fluorescence value, make a standard curve, the X axis is the H2O2 concentration, the y axis is the fluorescence value, and get an equation. Look at the fluorescence value of your sample, that is, the Y value, and the corresponding X value Yes.

9. After some cells are loaded with probes, the cells are easy to float up, and the experimental group will suck some of the cells when washing the cells. Therefore, the cell mass is doubled when the cells are planted, so that the cells are tightly connected and adhere to the wall more firmly, and the fluorescence value of the experimental group is higher. In addition, H2DCFDA is very sensitive. The concentration of the working solution should be lower, 1-2μM is enough. If the concentration is too high, non-specific staining may occur. This probe is very unstable. Once it is oxidized, the background fluorescence value will increase. It is best to use the working solution and prepare it.

Instructions for use

1. Load ROS probe

1.1 In-situ loading of probes (only for adherent cells)

a) Cell preparation: Plating cells the day before the test to ensure that the number of cells during the test is less than 5×10^5/ml.

b) Drug induction: Remove the cell culture medium, add the drug treatment diluted with serum-free medium, and incubate in a 37°C cell culture incubator in the dark. The actual induction time is determined by the characteristics of the drug and the cell type.

c) (Optional) Positive control: First dilute the positive control (Rosup, 100 mM) with serum-free medium, etc. to a common working concentration of 100 μM, add cells, and incubate at 37°C for 0.5 to 4 h in the dark to increase the level of reactive oxygen species , Different cell types are different. For example: HeLa cells need to be incubated for 30-60 minutes, and MRC5 human embryonic fibroblasts need to be incubated for 90 minutes.

d) ROS probe preparation: Before loading the probe, dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to make the final concentration 10 μM.

e) ROS probe loading: aspirate the treatment drug, and add DCFH-DA working solution diluted in an appropriate volume. The added volume must cover the cells sufficiently. For example, a 6-well plate is usually no less than 1000 μL, and a 96-well plate is usually no less than 100 μL. Incubate for 30 min in a cell culture box at 37°C in the dark.

f) Cell washing: Wash the cells with serum-free culture medium 1 to 2 times to fully remove the DCFH-DA that has not entered the cells.

1.2 Load probes after collecting cells (applicable to adherent cells and suspension cells)

a) Cell preparation: Cells are cultured according to standard methods, and the cell status for testing must be guaranteed. According to the appropriate method, wash and collect enough cells.

b) Drug induction: Suspend the collected cells in an appropriate amount of diluted drug, and incubate in a 37°C cell incubator in the dark. The actual induction time is determined by the characteristics of the drug and the cell type.

c) (Optional) Positive control: First dilute the positive control (Rosup, 100 mM) with serum-free medium to a common working concentration of 100 μM, add cells, and incubate at 37°C in the dark for 0.5 to 4 hours to increase the level of reactive oxygen species. There are differences in cell types. For example: HeLa cells need to be incubated for 30-60 minutes, and MRC5 human embryonic fibroblasts need to be incubated for 90 minutes.

d) ROS probe preparation: Before loading the probe, dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to make the final concentration 10 μM.

e) Probe loading: remove the intracellular drugs, collect the cells by centrifugation, and add the diluted probe to make the cell density 1×10^6~2×10^7.

Note: The cell density needs to be adjusted according to the subsequent detection system, detection method, and total detection volume. For example, for flow cytometry, the number of cells in a single tube is not less than 10^4 and not more than 10^6.

f) Cell washing: Wash the cells with serum-free cell culture medium 1-2 times to fully remove the DCFH-DA that has not entered the cells.

2. Fluorescence microscopy operation method

a) For adherent growth cells or living tissues, you can directly observe under a fluorescence microscope; for suspended growth cells, drop 25-50 μL of cell suspension onto a microscope slide, and then cover with a cover glass.

b) Under a fluorescence microscope, use FITC filters to observe fluorescence, and remove background to observe changes in fluorescence.

3. Flow cytometry operation method

a) For adherent growth cells, trypsinization is used to prepare a single cell suspension; for suspension growth cells, the cells are directly collected. Resuspend the cells with 0.5-1 mL PBS (0.5-1 x 10^5/ml).

b) Choose the FL1 or BL1 channel of the flow cytometer, excite at 488nm, and measure the emission at 530nm. Cells should be divided into two subgroups: ROS negative cells have only very low fluorescence intensity, and ROS positive cells have strong green fluorescence.

4. Parameter setting

Using 488nm excitation wavelength, 525nm emission wavelength, real-time or time-by-time detection of fluorescence intensity before and after stimulation. The fluorescence spectrum of DCF is very similar to FITC, and the parameters of FITC can be used to detect DCF. Refer to the figure below for the excitation and emission spectra of DCF.

Reactive oxygen species assay kit was used to display the fluorescence of reactive oxygen species in CHO cells.

Left image: CHO cells were treated with the reactive oxygen species included in the kit; right image: normal CHO cells. Green fluorescence indicates a sharp increase in cellular reactive oxygen species and can show its location.

Storage and Shipping

Storage

Protected from light,Store at -20°C

Shipped In

Ice chest + Ice pads

References

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