Application

This kit can be used to measure Reactive Oxygen Species (ROS) in tissue and cell samples.

Detection significance

Reactive oxygen species (ROS) are active chemical substances produced in the metabolic process of the body, including oxygen free radicals, hydrogen peroxide and its downstream products, such as peroxides and hydroxides. ROS are both necessary and harmful to organisms, and are involved in cell growth, proliferation, development and differentiation, aging and apoptosis, as well as many physiological and pathological processes. Excessive ROS will lead to oxidative stress and oxidative damage of cells, further promoting the occurrence and development of many diseases, such as cancer, cardiovascular disease and diabetes.

Detection principle

DCFH-DA (2,7-dichlorofuorescin diacetate) is a fluorescent probe without fluorescence that can freely cross the membrane. After entering the cell, it can be hydrolyzed by intracellular esterase to form DCFH (dichlorofluorescin). In the presence of reactive oxygen species (ROS), DCFH is oxidized to DCF (dichlorofluorescein) which is a strong green fluorescent substance that cannot penetrate the cell membrane. DCF has a maximum wave peak near the excitation wavelength of 502 nm and the emission wavelength of 525 nm, and the intensity is proportional to the level of intracellular reactive oxygen species.

Experimental instruments

Test tubes, Vortex Mixer, Centrifuge, Fluorescence Microplate reader (Ex/Em=500 nm/525 nm) Fluorescence Spectrophotometry (Ex/Em=500 nm/525 nm), Flow Cytometry (Ex/Em=500 nm/525 nm)

Operation steps

1. Detection of culture cell sample

(1) Add the fluorescent probe:

a. Add Reagent 1 working solution to the cells. The DCFH-DA working concentration can be 0.1-20 μM for different cells and treatment. Pre-experiment is suggested to determine the appropriate concentration. The total dilution ratio should be more than 1:500-1:1000 in order to avoid effects of DMSO on cells. DMSO should be set as solution control.

b. Incubate at 37℃ for 30 min ~ few hours, generally 30~60 min. The incubation time is related to cell types, stimulation conditions, and DCFH-DA concentration.

c. Cell collection:

Suspension cells: centrifuge the sample at 1000 g for 5~10 min and wash with Reagent 3 working solution for 2~3 times. Centrifuge and collect the cell precipitation for fluorescence detection.

Adherent cells: digest the cells with 0.25% trypsin, add medium to terminate the digestion, thus to prepare the cell suspension. Centrifuge at 1000 g for 5~10 min and collect cells, then wash with PBS for 1~2 times. Centrifuge and collect cell precipitation for fluorescence detection.

(2) Fluorescence detection:

a. Re-suspend collected cells with Reagent 3 working solution for detection.

b. Wavelength: the excitation wavelength is 500 nm, the emission wavelength is 525 nm. It can also be detected according to the fluorescence detection conditions of FITC.

Notes: the density of re-suspension cell is determined by cell fluorescence intensity. If fluorescence is strong (weak), then decrease (increase) the cell density, but cell density of all samples should be consistent.

2. Detection of tissue sample

(1) Preparation of single cell suspension:

Method 1: using the single cell suspension instrument.

Method 2: enzyme digestion.

a. Take the tissue into pre-cooled Reagent 3 working solution immediately and clean the blood and other contaminants. Remove the massive composition, fiber, fat, and blood vessels (except for specialized cells).

b. Cut the tissue into about 1 mm3 pieces with the ophthalmic scissors, then put these pieces to pre-cooled Reagent 3 working solution to remove the cell debris.

c. Add an appropriate amount of enzyme digestion, incubate in 37℃ water bath for 20~30 min and gently oscillate the mixture intermittently.

d. Stop the digestion with Reagent 3 working solution. Filter the mixture to remove the tissue massive component with nylon mesh (300 mesh) and collect the cells. Centrifuge at 500 g for 10 min and discard the supernatant, then wash with Reagent 3 working solution for 1~2 times. Re-suspend to prepare the single cell suspension solution. The cell amount should be no less than 106.

Method 3: mechanical method.

a. The pretreatment is the same as step a and step b in the enzyme digestion method.

b.Tight the nylon mesh (300 mesh) on a small beaker, then place the tissue pieces on the mesh and gently rub the tissue with ophthalmic scissor or erasing knife. Wash the tissue with Reagent 3 working solution at the same time.

c. Collect the cell suspension and centrifuge at 500 g for 10 min. Then discard the supernatant and wash with PBS for 1~2 times. Re-suspend to prepare the single cell suspension solution. The cell amount should be no less than 106.

(2) Add the fluorescent probe:

a. Add Reagent 1 working solution to the cells. The DCFH-DA working concentration can be 0.1-20 μM for different cells and treatment. Pre-experiment is suggested to determine the appropriate concentration. The total dilution ratio should be more than 1:500-1:1000 in order to avoid effects of DMSO on cells. DMSO should be set as solution control.

b. Incubate at 37℃ for 30 min ~ few hours, generally 30~60 min. The incubation time is related to cell types, stimulation conditions, and DCFH-DA concentration.

c. Collect the incubated single cell suspension, centrifuge at 1000 g for 5~10 min to collect cells. Wash with Reagent 3 working solution for 1~2 times. Centrifuge and collect the cell precipitation for fluorescence detection.

(3) Fluorescence detection:

a. Re-suspend collected cells with Reagent 3 working solution for detection.

b. Wavelength: the excitation wavelength is 500 nm, the emission wavelength is 525 nm. It can also be detected according to the fluorescence detection conditions of FITC.

Notes

1. The kit is for scientific research only.

2.  Instructions should be followed strictly, changes of operation may result in unreliable results.

3.  The validity of kit is 6 months.

4. Do not use components from different batches of kit.

5. The density of re-suspension cell is determined by cell fluorescence intensity. If fluorescence is strong (weak), then decrease (increase) the cell density, but cell density of all samples should be consistent.

6. Fluorescent substances are sensitive to light and should avoid light during detection.

7. DCF is easy to be quenched, and the samples after incubation must be detected within 2 hours.

8. Results were expressed as fluorescence intensity or geometric average fluorescence intensity (flow cytometry).

9. When using flow cytometry, in order to avoid the interference of cell debris and dead cells on the experimental results, it is necessary to eliminate them.

10. Set a positive control (Reagent 2 working solution) and a negative control (only cells without reagent 1 working solution).