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Instruction manual for reactive oxygen species (ROS) determination kits

 Last Update: 2022-09-14
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This kit is only intended for scientific research and laboratory use

Active Oxygen Species (ROS) Assay Kit Instructions

(SKU:E004-1-1 Chemical Fluorescence 100T-500T)

First, the significance of the measurement:

Reactive oxygen species (ROS) include superoxide radicals, hydrogen peroxide, and their downstream products, peroxides and hydroxylides, which are involved in cell growth and proliferation, developmental differentiation, aging and apoptosis, and many physiological and pathological processes
.
The DCFH-DA (2,7-Dichlorofuorescin Diacetate) probe used in this kit is the most commonly used and sensitive intracellular reactive oxygen species detection probe
to date.
DCFH-DA itself has no fluorescence and can freely cross the cell membrane, and when it enters the cell, it is hydrolyzed to DCFH (Dichlorofluorescin)
by the associated esterase in the cell.
DCFH, on the other hand, does not penetrate the cell membrane, making it easy for the probe to be labeled inside the
cell.
When reactive oxygen species are present in cells, DCFH is oxidized to a strong green fluorescent substance DCF (Dichlorofluorescein), whose fluorescence has a maximum peak near the excitation wavelength of 488 nm and an emission wavelength of 525 nm, and its fluorescence intensity is proportional to the level of reactive oxygen species in the cell
.
In general, the elevation of reactive oxygen species in cells is one of the hallmarks of apoptosis
.
The reactive oxygen species detection system has a low background, high sensitivity, good repeatability and easy operation
.

Operating wavelength: Optimal excitation wavelength 488, optimal emission wavelength 525 (53020 nm).
It can also be detected according to FITC fluorescence detection
conditions.

Second, the composition and preservation of reagents:

1, 0.
1mL 10mM DCFH-DA in DMSO, 4-8 °C storage
.

2.
1mL reactive oxygen species positive control (Rousp, 50mg/mL), stored
at 4-8 °C.

Third, reagent preparation:

1.
DCFH-DA can be diluted in a suitable buffer, such as serum-free culture solution or 0.
01M PBS
.
For different treatment steps, the working concentration of DCFH-DA can be 100 nM to 20 μM, and pre-experiments are required to determine the appropriate concentration
.
The overall dilution should be above 1:500 to 1:1000 to avoid the effects of DMSO on cells, and the recommended initial concentration is 10 μM
.
In addition, for some cells, if no stimulus-negative control cell fluorescence is also found to be strong, DCFH-DA can be diluted at 1:2000 to 5000 so that the concentration of DCFH-DA when loading the probe is 2 to 5 μmol/L
.
The probe loading time can also be adjusted
within 15 to 60 minutes depending on the situation.

2.
The positive control can be used
in the ratio of 1:1000.
For example, a total of 1 mL of cells loaded with probes can be added to a positive control stimulus of 1 mL (a very significant increase in reactive oxygen species levels can usually be observed within 20 to 30 minutes of stimulation
).
For different cells
.
The effect of reactive oxygen species controls may vary considerably
.
If the increase in reactive oxygen species is not observed within 30 minutes after stimulation, the concentration of the reactive oxygen positive control can be appropriately increased, and conversely, if the reactive oxygen species rises too quickly, its concentration
can be appropriately reduced.
Reactive oxygen positive controls are only used as positive controls for samples, and not reactive oxygen positive controls
need to be added to every sample.
If the user is familiar with ROS fluorescence or if a positive control tube is not necessary for the experiment, such as fluorescence microscopy detection, the reagent
can also be absent.

IV.
Operation steps of tissue samples:
(Can be observed by laser confocal microscopy, and can also be used for flow cytometry, fluorescence microplate reader, fluorescence spectrophotometer determination)

1.
Preparation of single cell suspension:

Method 1: Single cell suspension was prepared by single cell suspension
preparation instrument.

Method 2, enzyme digestion:

(1) Immediately put the selected tissue into the pre-chilled tissue culture solution or PBS to clean the blood stains and other contaminants
.
Remove dead components, fibers, fats, and blood vessels from tissue blocks (except when cells are specially prepared
).

(2) Use ophthalmic scissors to cut the tissue blocks into small pieces of about 1 mm3, place them in the pre-chilled tissue culture solution or PBS and rinse them to wash off the shredded cell fragments
.

(3) Add an appropriate amount of enzyme digestion solution, digest at 37 °C in a constant temperature water bath for 20 to 30 minutes, and perform intermittent shaking or blowing of cells
during the period.

(4) Terminate the digestion with tissue culture medium or PBS, remove the tissue mass by filtering with a 300 mesh nylon mesh, collect the filtered cells, centrifuge 500g for 10 min, leave the supernatant, and wash 1 to 2 times
with PBS.

Method 3, mechanical method (net rubbing method):

(1) Pre-treatment homoenzyme digestion (1), (2) steps
.

(2) Tie the 300 mesh nylon mesh to a small beaker, place the shredded tissue on the net, gently rub the tissue block with ophthalmic tweezers or spatula, and rinse with PBS while rubbing until the tissue is finished
.

(3) Collect the cell suspension, centrifuge 500g for 10 min, leave the supernatant and leave the pellet, and wash 1 to 2 times
with PBS.

2.
Add fluorescent probes:

(1) Take the cells that are not treated with 0.
01M

PBS resuspended and set to a negative control tube
.
Positive control tube: Resuspend the cell pellet with diluted DCFH-DA while adding reactive oxygen species to the hydrogen body to induce the cells
.

(2) Sample tube: resuspend the cell pellet with diluted DCFH-DA, and the cell density generally requires 1×106-2×107/mL
.

(3) Incubate the cells at 37 °C for 30min to a few hours, and mix them upside down every 3-5 minutes to make the probe in full contact
with the cells.
Usually 20 to 60 minutes is sufficient, and the length of incubation is related
to cell type, stimulation conditions, and DCFH-DA concentration.

(4) Collect the single cell suspension after incubation (probe labeling), 1000g, centrifuge for 5 to 10 minutes, remove the supernatant to collect the cell pellet, and wash 1 to 2 times with PBS to fully remove the DCFH-DA
that has not entered the cell.
Centrifugation to collect cell pellets for fluorescence detection;

3.
Fluorescence detection: This kit is only used for scientific research and laboratory

(1) Resuspend the above collected cell pellet with PBS and use it for detection;

(2) Wavelength setting: the best excitation wavelength is 488 nm, and the best emission wavelength is 525 nm
.
It can also be detected under FITC fluorescence detection
conditions.

(3) The results are expressed
as fluorescence values.

V.
Cell sample operation steps:
(Can be observed by laser confocal microscopy, and can also be used for flow cytometry, fluorescence microplate reader, fluorescence spectrophotometer determination)

1.
Add the probe directly to the culture medium (this method is only suitable for adherent cells):

(1) Directly add the DCFH-DA probe to the serum-free medium: generally dilute DCFH-DA with serum-free culture solution at 1:1000 (final concentration of 10 μM).

After removing the culture medium, add the appropriate volume of diluted DCFH-DA
.
The added volume is appropriate to adequately cover the cells, usually adding a diluted DCFH-DA of not less than 1 mL to one well of a 6-well
plate.

(2) Take a copy of the cells without adding probes and only adding medium to the negative control tube
.
Positive control tube: Take a copy of the cells that have been added to the probe and add the reactive oxygen species to the hydrogen-induced cells
.

(3), 37 °C incubation of cells for 20min to a few hours (every 3 to 5 minutes upside down mixing, so that the probe and the cells are in full contact), usually 20min, the length of incubation time is related
to cell type, stimulation conditions, DCFH-DA concentration.
Typically, a positive control observes significant green fluorescence
after stimulating cells for 20 to 30 minutes.

(4) Aspirate the culture medium, use serum-free culture solution or 0.
01MPBS to repeatedly blow, the naked eye observes the bottom of the bottle from translucent (single layer of cells connected into pieces) to transparent, and the cell layer is almost all blown into PBS
.

(5) Collect all the cell suspensions into a 1.
5 mL centrifuge tube
.
Wash 2 times with serum-free culture medium or PBS to adequately remove DCFH-DA
that has not entered the cell.
1000 rpm/min, 5 min, add PBS resuspensive cells for assay after aspirate supernatant
.

(6) Wavelength setting: the best excitation wavelength is 488 nm, and the best emission wavelength is 525 nm
.
It can also be detected according to FITC fluorescence detection
conditions.

(7) The result is expressed
as a fluorescence value.

2.
Collect cells first, prepare a cell suspension and then determine it (this method is suitable for adherent cells and suspension cells)

(1) Cell collection:

a.
Adhere to the cells, aspirate the culture medium, use the serum-free culture medium or 0.
01MPBS to repeatedly blow, the naked eye observes the bottom of the well plate (bottle bottom) from translucent (cell single layer connected into pieces) to transparent, the cell layer is almost all blown into PBS
.

Collect all the cell suspensions into a 1.
5 mL centrifuge tube
.
Wash 2 times with serum-free culture medium or 0.
01MPBS at 1000 rpm/min, centrifuge for 5 min, aspirate the supernatant, leaving the cell pellet for the assay
.

c.
Centrifuge the suspended cells according to the conventional method (2000 rpm/min, centrifuge for 5 min), collect the cell pellet, wash twice with serum-free culture solution or 0.
01M PBS, centrifuge for 5 min, aspirate the supernatant, and leave the cell pellet for assay
.

(2), cell resuspension: cell density generally requires 1×106-2×107/mL, generally there are two methods:

a.
First add serum-free culture solution or PBS resuspend the cells, and then add probes according to the initial concentration of 10 μM according to the volume of added culture medium or PBS (it is best to do pre-experiments to determine the appropriate concentration of your own samples), (suitable for pre-experiments and small samples)

b.
First dilute the probe with serum-free culture solution or PBS according to the concentration of 10 μM, and then resuspend the above cell pellet with the diluted probe to prepare a cell suspension, (suitable for cases with more samples)

(3) Take one portion of the cells without a probe and only add medium or PBS as a negative control tube
.
Positive control tube: Take a portion of the cell suspension that has been added to the probe, and add the reactive oxygen species hydrogen body to induce the cells
.

(4) Incubate cells at 37 °C for 20min to a few hours, usually 20 to 60min, and the incubation time is related to cell type, stimulation conditions, and DCFH-DA concentration; Mix upside down every 3-5 minutes to bring the probe into full contact
with the cells.

(5) Collect the single cell suspension after incubation (probe labeling), 1000 rpm/min, centrifuge for 5 min, aspirate the supernatant, wash 1 to 2 times with PBS, centrifuge to collect the cell pellet for fluorescence detection
.

(6) Resuspend the above collected cell pellet with PBS and use it for detection
.

(7) Wavelength setting: the best excitation wavelength is 488nm, and the best emission wavelength is 525nm
.
It can also be detected according to FITC fluorescence detection
conditions.

(8) The results are expressed
as fluorescence values.

6.
Relevant matters needing attention

1.
After the suspension fluorescent probe is labeled, be sure to wash the remaining probe that has not entered the cell, otherwise it will cause the background to be unclean and the fluorescence value will be high
.

2.
The cell density of the resuspending can be adjusted according to the fluorescence strength of the cells, such as the cell density can be adjusted to be smaller if the fluorescence is stronger, and the cell density can be increased if the fluorescence is weak, but the cell density of all samples should be consistent
.

3.
When measuring the cell sample, for the cells with a drug action time of less than 2 hours, it is generally recommended to label the probe first, and then stimulate the cells with the drug, for the cells with a drug action time of more than 6 hours, the cells can be stimulated with reactive oxygen positive control and drugs first, and then label the probe
.

4.
At the same time, part of the single cell suspension is taken by ultrasound or homogenization treatment for the determination of protein (protein quantification kit is sold in this book, recommended A045-2 Coomassie bright blue protein quantitative kit, A045-3 BCA protein quantitative kit), for the result calculation is expressed as fluorescence value / mg protein
.

5, fluorescence is generally the intensity of fluorescence emission, but because different instruments are different in representation methods, some use light energy, some use photons to count, there is no comparability between the measurement values of different instruments, generally expressed
by relative values.
It is therefore an Arbitrary Unit (AU
).

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