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Click-iT EdU-594 Cell Proliferation Detection Kit

Volume: 100T

  • G1603
  • Servicebio

Product Description

Product introduction:

 

It is a common and important assessment method in the life sciences to determine the influence of certain genes, drugs, etc. on the cells cultured in vitro by analyzing the cell proliferation ability, or analyzing the growth and renewal ability of individual tissue cells under different conditions or stimulation interventions. . There are many methods for detecting cell proliferation. Most of them use some metabolic enzymes produced by cells to indirectly assess the proliferation activity of cells (such as CCK-8 method, MTT method, etc.), but some drugs or factors such as the state of the cell itself will Have a certain impact on the results of the assessment. Direct detection of DNA synthesis in cells to determine cell proliferation is recognized as the most accurate and effective detection method. However, both the original radiolabeled nucleoside incorporation method and the subsequent improvement of the BrdU method based on antibody detection have their own limitations.

 

EdU (5-Ethynyl-2'-deoxyuridine, 5-ethynyl-2'-deoxyuridine) is a thymidine analogue containing an acetylene group, when injected into animals or Incubating cells cultured in vitro, these small molecules can quickly diffuse to various organs and tissues, and infiltrate into cells, and can replace thymidine (T) into newly synthesized DNA during cell proliferation. The acetylene group in the EdU molecule can react with the fluorescently labeled azide compound probe to form a stable triazole ring under the catalysis of copper ions, so the newly synthesized DNA can be labeled with the corresponding fluorescent probe. Compared with the radiolabeled nucleoside incorporation method, the EdU detection method has no limiting factors such as radioactive contamination; compared with the BrdU detection method, the EdU detection method does not require DNA denaturation treatment or antigen-antibody reaction, which greatly reduces the complexity of the experiment And time, it also becomes more time-saving, more sensitive, more stable and more accurate. This kit can be used to detect cell proliferation in cultured cells or animal tissues in vitro. The fluorescent probe in this kit is red fluorescence, the maximum excitation wavelength is 593 nm, and the maximum emission wavelength is 614 nm. After the proliferating cells are labeled, the cell nucleus will show bright red fluorescence. The cell nucleus is jointly labeled with the matching conventional nuclear dye (This kit provides Hoechst 33342 cell nuclear dye), you can use fluorescence microscope, laser confocal microscope and other instruments to directly observe cell proliferation; you can also use flow cytometry to detect the fluorescence intensity of cultured cells in vitro, and judge the cell cycle based on the fluorescence intensity DNA replication activity in the mid-S phase.

 

Storage and transportation

 

Transported in wet ice; stored at -20°C in the dark, EdU catalytic reagent (reagent A) and reaction buffer can be stored at 4°C; valid for 12 months.

 

Component Number

Component

G1603-100T

G1603-1

EdU Storage solution(10 mM)

100 μL

G1603-2

Catalytic reagent (reagent A)

120 μL

G1603-3

Fluorescent dye iF488 (reagent B)

50 μL

G1603-4

Catalytic additives (reagent C)

2×100 mg(powder)

G1603-5

Reaction Buffer

20 mL

G1603-6

Hoechst 33342 Staining Solution

30 μL

Product User Manual

1pc

 

Note: The above reaction times correspond to 96-well plate detection.

 

Experiment preparation

 

1. Cell culture medium containing serum;

 

2. Permeabilization solution: buffer solution containing 0.2-0.5% Triton X-100 (G1204 is recommended);

 

3. Fixing solution: 4% paraformaldehyde (G1101 recommended) or other reagents for similar purposes;

 

4. PBS buffer (G4202 is recommended);

 

5. Ultra-pure water;

 

6. Animal modeling and tissue section related reagents (animal tissue cell proliferation detection).

 

Operation Steps:

 

1. Preparation of in vitro cell samples and reagents:

 

1.1. Plant the cells evenly in the cell culture plate at a certain density (the planting density is determined by factors such as cell size, growth speed, etc.). After the cells adhere to the wall or return to a normal state, perform corresponding drug stimulation and other treatments (such as testing suspension Cells, please follow the normal operation method of suspension cells, the whole experiment needs to add steps such as centrifugation).

 

1.2. Centrifuge the catalytic additive (reagent C) at low speed, add 1 mL of ultrapure water to dissolve it, and store it at -20℃ for later use.

 

2. EdU labeling, fixation and permeabilization of cells in vitro:

 

2.1. Prepare 2×EdU incubation working solution: add 2 μL EdU storage solution (10 mM) to every 1 mL of complete cell culture medium, which is 20 μM 2×EdU incubation working solution, and put it in the incubator to preheat ( It is recommended that the pre-experiment be explored with EdU working concentration of 10 μM);

 

2.2. In the mode of half-changing medium, aspirate half of the original cell culture medium in the culture plate, and add an equal volume of preheated 2×EdU incubation working solution, and incubate for a certain period of time (the incubation time generally depends on the corresponding cell growth The cycle usually accounts for about 10% of the cell cycle time. For most adherent and fast-growing cells, it is recommended to incubate for about 2 hours. For specific conditions, it needs to be adjusted according to the cell characteristics and actual conditions after treatment. If longer incubation is required Time can appropriately reduce EdU working concentration; for a shorter time, EdU concentration can be appropriately increased);

 

2.3. After the EdU-labeled cell sample is washed with PBS buffer for 1-2 times, add the fixative solution to cover the cells, and fix at room temperature for 15 minutes (if flow detection is required, adhere to the cells before this step After digestion and resuspension, fix it, and then follow the suspension cell processing method); Wash 2-3 times with PBS buffer, each time for 3-5 min;

 

2.4. Remove the PBS buffer, add permeate to cover the cells or tissues, and incubate at room temperature for 15 minutes;

 

2.5. After removing the permeate, wash 1-2 times with PBS buffer for 3-5 min each time, and then go to step 4.

 

3. Animal EdU injection modeling and tissue section processing:

 

3.1. According to the experimental requirements, one or more EdU injections are used to model animals by intraperitoneal injection, intramuscular injection, subcutaneous injection, tail vein injection, etc. Generally, the ratio of EdU dosage to animal body weight is 5 mg/kg. Specific injection The amount depends on the research content and animal conditions. Part of EdU storage solution is provided in this kit, which is mainly used for in vitro cell EdU labeling. If you need to model animals with EdU, you can order EdU reagent separately (G5059 is recommended);

 

3.2. Small intestine and other epithelial tissue cells proliferate fast, and brain tissue cells proliferate slowly. The faster-growing tissues usually take less than 12 hours to mark, while the slower-growing tissue may take several days to mark; the best marking time is based on Depending on the specific experiment, because the small intestinal epithelial tissue proliferates quickly, it is recommended to use this type of tissue as a label reference;

 

3.3. After the modeled animals are put to death according to the prescribed standards, the required tissues are taken out, and frozen sections or paraffin sections are made according to conventional procedures;

 

a. For frozen sections: return to room temperature, add an appropriate amount of fixative, and fix at room temperature for 15 minutes. Remove the fixative and wash 3 times with an appropriate amount of PBS buffer for 3-5 min each; remove the PBS buffer and cover the tissue with an appropriate amount of permeabilization solution and incubate at room temperature for 10-15 min; remove the permeabilization solution and wash 1 with PBS buffer -2 times, 3-5 min each time, then go to step 4.

 

b. For paraffin sections: deparaffinize and rehydrate the sections and wash with PBS for 5 min. Remove the PBS buffer, add permeate to cover the cells or tissues, and incubate at room temperature for 15 min; after removing the permeate, wash 1-2 times with PBS buffer for 3-5 min each time, and then go to step 4.

 

4. EdU click response:

 

4.1. During cell or tissue fixation and perforation, prepare the click reaction solution (for different sample preparation systems, refer to the scheme in the table below)

 

For cells cultured in vitro: This reference step corresponds to the volume and dosage of 10 96-well plate samples (100 μL per well). The preparation amount can be increased or decreased in proportion to the requirements of use. Please add the components in the order shown in the table below. Add side and mix well (prepared and used now);

 

Component

Volume

Reaction Buffer

935 μL

Catalytic Reagent (reagent A)

10 μL

Fluorescent DYE iF594 (reagent B)

5 μL

Catalytic Additives (reagent C)

50 μL

Total Capacity

1000 μL

 

For tissue cell slices: refer to the following system for the preparation of the click reaction solution system. The preparation amount can be increased or reduced in proportion to the number of cut samples. Each slice sample is covered with about 100-200 μL of click reaction solution.

 

Component

Volume

Reaction Buffer

928 μL

Catalytic Reagent (reagent A)

10 μL

Fluorescent DYE IF594 (reagent B)

12 μL

Catalytic Additives (reagent C)

50 μL

Total Capacity

1000 μL

 

4.2. Remove the PBS buffer of the previous step (step 2.5 or 3.3), add the click reaction solution, shake gently to ensure that the reaction solution covers all the cells or tissues, and incubate for 30 min at room temperature in the dark;

 

4.3. Remove the click reaction solution and wash 2-3 times with PBS buffer for 3-5 min each time (if there are no other special requirements, you can use flow cytometry to detect the fluorescence intensity or use other instruments to detect the fluorescence effect).

 

5. Nuclear staining:

 

5.1. Remove the PBS buffer in the previous step, dilute the Hoechst 33342 staining solution and PBS buffer at a ratio of 1:500-1000, add the covering cells, and incubate for 5 min;

 

5.2. Remove the Hoechst 33342 staining solution, and wash 2-3 times with PBS buffer for 3-5 min each time.

 

6. Imaging and detection analysis

 

Directly place the cultured cells or tissue slice samples in a fluorescence microscope or a confocal microscope to analyze the proportion of proliferating cells; or collect the cells cultured in vitro and use a flow cytometer to detect the fluorescence intensity (it is recommended to use a The EdU-labeled cell sample is used as a negative control for flow cytometry detection, and the appropriate voltage is selected). According to the fluorescence intensity, the DNA replication activity in the S phase of the cell cycle can be judged. The corresponding spectral characteristics of the fluorescent dye iF594 (reagent B) of this kit are Ex/Em: 593 nm/614 nm (red); the corresponding spectral characteristics of Hoechst 33342 staining solution are Ex/Em: 346 nm/460 nm (blue) .

 

Notes:

 

1. For cells cultured in vitro, the specific EdU concentration and incubation time can be adjusted appropriately depending on the sample and the purpose of the research.

 

2. Some tissue cells proliferate slowly. In order to eliminate factors such as poor modeling effects, it is recommended to select tissue samples with fast proliferation as reference samples (such as intestinal tissue).

 

3. If the background color is too dark, it may be caused by insufficient washing in the experiment, too long tissue sample fixation time, and residual fixative.

 

4. EdU catalytic addition reagent (reagent C) is easy to oxidize. Try to avoid prolonged exposure to the air. After being prepared into an aqueous solution, it is recommended to store in aliquot; after testing, if the color of EdU catalytic addition reagent changes slightly, the click reaction catalytic system remains the same It can proceed normally. If it appears brown, it indicates that the component has expired, so please discard it.

 

5. Please wear lab coat and disposable gloves during operation.

 

This product is for scientific research purposes only, not for clinical diagnosis!

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