Click-iT EdU-594 Cell Proliferation Assay Kit 100T

Product Information

Introduction

It is a common and important evaluation method in life sciences to judge the influence of certain genes, drugs, etc. on cells cultured in vitro by analyzing the cell proliferation ability, or to analyze the growth and renewal ability of individual tissue cells under different states or stimulation interventions. . At present, 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 the state of cells themselves will have a certain impact on the evaluation results. Direct detection of DNA synthesis in cells to determine cell proliferation is recognized as the most accurate and effective detection method. However, both the initially used radiolabeled nucleoside incorporation method and the subsequent improved BrdU method based on antibody detection have certain limitations.

EdU (5-ethynyl-2'-deoxyuridine) is a thymidine analog containing an acetylene group that when injected into animals or incubated with cells in vitro, these Small molecules can rapidly diffuse into various organs and tissues, penetrate into cells, and can be incorporated into newly synthesized DNA in place of thymidine (T) during cell proliferation. The acetylene group in the EdU molecule can undergo a "click" reaction with the fluorescently labeled azide probe under the catalysis of copper ions to form a stable triazole ring, 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 limitations such as radioactive contamination; compared with the BrdU detection method, the EdU detection method does not require DNA denaturation treatment, nor does it require antigen-antibody reaction, which greatly reduces the complexity of the experiment and time, and makes it more time-efficient, more responsive, more stable and more accurate. This kit can be used to detect cell proliferation in cells cultured in vitro or in animal tissues. The fluorescent probe in this kit is red fluorescence with a maximum excitation wavelength of 593 nm and a maximum emission wavelength of 614 nm. After the proliferating cells are labeled, the nucleus will show bright red fluorescence, and the nuclei will be labeled with a conventional nuclear dye (Hoechst 33342 nuclear dye is provided in this kit). The fluorescence intensity of the in vitro cultured cell population can also be detected by flow cytometry, and the DNA replication activity in the S phase of the cell cycle can be judged according to the fluorescence intensity.

Storage and Handling Conditions

Transport with wet ice; store at -20°C in the dark. EdU catalytic reagent (reagent A) and reaction buffer can be stored at 4°C; the validity period is 12 months.

Components

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

Experiment preparation

1. Serum-containing cell culture medium;

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

3. Fixative: 4% paraformaldehyde (recommended G1101) or other similar reagents;

4. PBS buffer (recommended G4202);

5. Ultrapure water;

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

Steps

1. In vitro cell samples and reagent preparation:

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

1.2. The catalytic additive (reagent C) was centrifuged at low speed, dissolved in 1 mL of ultrapure water, and stored at -20°C 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 stock 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 an incubator to preheat ( It is recommended to explore with EdU working concentration of 10 μM in preliminary experiments);

2.2. In the mode of half medium exchange, half of the original cell culture medium in the culture plate was removed by suction, and an equal volume of pre-warmed 2×EdU incubation working solution was added for a certain period of time (the incubation time generally depends on the corresponding cell growth). The cell cycle usually accounts for about 10% of the cell cycle. For most adherent and fast-growing cells, it is recommended to incubate for about 2 hours. The specific situation needs to be adjusted according to the characteristics of the cells and the actual situation after treatment. If you need to incubate for a longer time The working concentration of EdU can be appropriately reduced for a short period of time; the concentration of EdU can be appropriately increased for a shorter period of time);

2.3. Wash the EdU-labeled and incubated cell samples with PBS buffer for 1-2 times, add fixative to cover the cells, and fix them at room temperature for 15 minutes (if flow detection is required, attach cells before this step. Digest and resuspend and then fix, and then follow the treatment method of suspended cells); wash 2-3 times with PBS buffer, 3-5 min each time;

2.4. Remove PBS buffer, add permeabilization solution to cover cells or tissues, and incubate at room temperature for 15 min;

2.5. After removing the permeabilizer, wash 1-2 times with PBS buffer for 3-5 min each time, 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 were performed on animals by intraperitoneal injection, intramuscular injection, subcutaneous injection, and tail vein injection. The amount depends on the research content and animal conditions. This kit provides some EdU storage solutions, which are mainly used for EdU labeling of cells in vitro. If EdU modeling is required for animals, EdU reagents (recommended G5059) can be ordered separately;

3.2. Epithelial tissue cells such as the small intestine proliferate rapidly, and tissue cells such as the brain proliferate slowly. The fast-growing tissue parts are usually marked for less than 12 hours, while the slow-growing tissue may take several days to be marked; the best marking time is based on It depends on the specific experiment. Due to the rapid proliferation of small intestinal epithelial tissue, it is recommended to take this type of tissue as a marker reference;

3.3. After the model animals were sacrificed according to the prescribed standards, the required tissues were taken out, and frozen sections or paraffin sections were made according to routine 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 time; remove the PBS buffer, use an appropriate amount of permeabilization solution, and incubate at room temperature for 10-15 min; remove the permeabilization solution and wash with PBS buffer for 1- 2 times, 3-5 min each time, then go to step 4.

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

4. EdU click response:

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

For cells cultured in vitro: This reference step corresponds to the volume of 10 96-well plate samples (100 μL per well). The preparation volume can be increased or decreased in equal proportions according to the needs of use. Please add the components in the order shown in the table. Add edge and mix well (currently prepared and used);

For tissue and cell slices: Refer to the system below for the preparation of the click reaction solution. The preparation volume can be increased or decreased in equal proportions according to the number of cut samples. Each slice sample is covered with about 100-200 μL of click reaction solution.

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

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

5. Nuclei staining:

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

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

6. Imaging and detection analysis

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

Note

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

2. Some tissue cells proliferate slowly. In order to exclude factors such as poor modeling effect, 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, long fixation time of tissue samples, and residual fixative.

4. The EdU catalytic addition reagent (reagent C) is easy to be oxidized, try to avoid prolonged exposure to the air. After being prepared into an aqueous solution, it is recommended to store in separate packages; after testing, if the color of the EdU catalytic addition reagent changes slightly, the click reaction catalytic system remains the same. It can be carried out normally, if it turns brown, it indicates that the component has failed, please discard it.

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

For Research Use Only!