16. Click-iT EdU-555 Cell Proliferation Assay Kit. 100 T （EdU-555) $650; 200T $900

Product Introduction

Cell proliferation analysis is a common and important method used in life sciences to assess the effects of genes, drugs, and other factors on cultured cells, as well as to analyze the growth and renewal capacity of cells in biological tissues under different conditions or stimuli. Currently, there are many methods available to detect cell proliferation, most of which rely on the activity of metabolic enzymes produced by cells to indirectly assess cell proliferation (such as the CCK-8 assay, MTT assay, etc.). However, factors such as drugs or the cellular state itself can influence the results of these assays. Directly detecting DNA synthesis in cells is recognized as the most accurate and effective method for assessing cell proliferation. However, both the initial radioactive nucleoside incorporation method and the subsequent antibody-based BrdU method have their own limitations.

EdU (5-Ethynyl-2′-deoxyuridine) is a thymidine analog that contains an ethynyl group. When injected into animals or incubated with cells in vitro, these small molecules can quickly diffuse into various organs and tissues and penetrate into cells. During the period of cell proliferation, EdU can replace thymidine (T) and be incorporated into newly synthesized DNA. The ethynyl group in the EdU molecule can react with azide-labeled fluorescent probes through a “click” reaction catalyzed by copper ions, forming a stable triazole ring. This allows the newly synthesized DNA to be labeled by the corresponding fluorescent probe. Compared to the radioactive nucleoside incorporation method, the EdU detection method does not have limitations such as radioactive contamination. Compared to the BrdU detection method, the EdU detection method does not require denaturation of DNA or antigen-antibody reactions, which greatly reduces the complexity and time of the experiment, making it more time-saving, sensitive, stable, and accurate. This assay kit can be used to detect cell proliferation in cultured cells or animal tissues. The fluorescent probe in this kit emits red fluorescence, with a maximum excitation wavelength of 557 nm and a maximum emission wavelength of 570 nm. Cells undergoing proliferation will exhibit bright red fluorescence in the nucleus after being labeled. Cell nuclei can be co-labeled with a conventional nuclear stain (Hoechst 33342 nuclear stain provided in this kit). The proliferation of cells can be directly observed using fluorescence microscopy, confocal laser scanning microscopy, and other instruments. It can also be detected by flow cytometry to measure the fluorescence intensity of cell populations in culture, and the DNA replication activity during the S phase of the cell cycle can be determined based on the fluorescence intensity.

Storage and Transportation

Transportation on wet ice; store in the dark at -20°C. EdU catalytic reagent (Component A) and reaction buffer can be stored at 4°C. Shelf life is 12 months.

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

Experimental Preparation

1. Serum-containing cell culture medium.
2. Permeabilization solution: Buffer containing 0.2-0.5% Triton X-100 (recommended G1204).
3. Fixative solution: 4% paraformaldehyde (recommended G1101) or similar reagents.
4. PBS buffer (recommended G4202).
5. Ultrapure water.
6. Reagents related to animal modeling and tissue sectioning (for cell proliferation detection in animal tissues).

Procedure

1. Preparation of in vitro cell samples and reagents:

1.1. Seed cells evenly in cell culture plates at a certain density (the seeding density is determined by factors such as cell size and growth rate). After the cells adhere or recover to the normal state, perform corresponding drug stimulation or other treatments. (For suspension cells, follow the standard procedures for suspension cell handling, which may involve centrifugation steps throughout the experiment.)

1.2. Centrifuge the catalytic additive (reagent C) at low speed, dissolve 100 mg in 1 mL of ultrapure water, aliquot, and store at -20°C. The remaining powder can be kept as a backup.

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

2.1. Prepare 2× EdU incubation working solution: Add 2 μL of 10 mM EdU storage solution to 1 mL of complete cell culture medium, resulting in a 20 μM 2× EdU incubation working solution. Preheat the solution in a cell incubator (It is recommended to perform a pre-experiment to determine the optimal working concentration of EdU, typically around 10 μM).

2.2. Aspirate half of the original cell culture medium from the culture plates and replace it with an equal volume of preheated 2× EdU incubation working solution. Incubate for a certain period of time (The incubation time generally depends on the corresponding cell growth cycle and is usually about 10% of the cell cycle duration. For most adherent cells with fast growth, it is recommended to incubate for about 2 hours. Adjust the incubation time based on cell characteristics, post-treatment conditions, etc. If longer incubation time is required, the working concentration of EdU can be appropriately reduced; for shorter incubation time, it can be increased).

2.3. After incubation with EdU, wash the cell samples with PBS buffer 1-2 times and then add the fixative solution to cover the cells. Fix at room temperature for 15 minutes. (For flow cytometry detection, digest and resuspend adherent cells before fixation, then follow the procedures for handling suspension cells.) Wash 2-3 times with PBS buffer, 3-5 minutes per wash.

2.4. Remove the PBS buffer and add the permeabilization solution to cover the cells or tissues. Incubate at room temperature for 15 minutes.

2.5. After removing the permeabilization solution, wash the samples 1-2 times with PBS buffer, 3-5 minutes per wash, then proceed to step 4.

3. EdU injection for animal modeling and tissue sectioning:

3.1. According to the experimental requirements, perform one or multiple EdU injections on animals using methods such as intraperitoneal injection, intramuscular injection, subcutaneous injection, or tail vein injection. The recommended ratio of EdU dosage to animal body weight is 5 mg/kg. The specific injection volume depends on the research content and animal conditions. This kit provides a portion of EdU storage solution primarily for in vitro cell labeling. If EdU modeling is required for animals, EdU reagent (recommended G5059) can be purchased separately.

3.2. Tissues with fast cell proliferation rates, such as the small intestine, have shorter labeling times (usually less than 12 hours), while tissues with slower proliferation rates may require several days for labeling. The optimal labeling time depends on the specific experiment. Considering the fast proliferation rate of intestinal epithelial tissue, it is recommended to use this tissue as a reference for labeling time.

3.3. After euthanizing the animals according to the prescribed standards, extract the desired tissues and prepare frozen sections or paraffin sections following routine procedures.

a. For frozen sections: Allow the sections to reach room temperature, add an appropriate amount of fixative solution, and fix at room temperature for 15 minutes. Remove the fixative solution, wash with PBS buffer 3 times, 3-5 minutes per wash. Remove the PBS buffer, add the permeabilization solution to cover the tissue, and incubate at room temperature for 10-15 minutes. Remove the permeabilization solution, wash with PBS buffer 1-2 times, 3-5 minutes per wash, then proceed to step 4.

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

4. EdU click reaction:

4.1. During the fixation and permeabilization of cells or tissues, prepare the click reaction mixture (refer to the table below for different sample system formulations).

For in vitro cultured cells: The reference procedure corresponds to the volume required for 10 samples in a 96-well plate (100 μL per well). Adjust the formulation accordingly by proportion if needed. Add the components in the order specified in the table and mix thoroughly while adding (prepare and use immediately).

For tissue cell sections: Refer to the following system for the preparation of the click reaction mixture. The preparation volume can be increased or decreased proportionally based on the number of tissue samples. Approximately 100-200 μL of click reaction mixture should be used to cover each section.

4.2. Remove the previous PBS buffer (from step 2.5 or 3.3) and add the Click reaction solution. Gently shake to ensure that the reaction solution covers all cells or tissues. Incubate at room temperature in the dark for 30 minutes.

4.3. Remove the Click reaction solution and wash with PBS buffer 2-3 times, each time for 3-5 minutes (unless otherwise specified, the fluorescence intensity can be detected using a flow cytometer or other instruments).

5. Nuclear Staining:

5.1. Remove the previous PBS buffer and add Hoechst 33342 staining solution diluted with PBS buffer at a ratio of 1:500-1000. Cover the cells and incubate for 5 minutes.

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

6. Imaging and Detection Analysis

For in vitro cultured cells or tissue sections, directly place them in a fluorescence microscope or confocal microscope for detection and analyze the proportion of proliferating cells. Alternatively, collect the in vitro cultured cells and detect the fluorescence intensity using a flow cytometer (it is recommended to use unmarked cells as a negative control for flow cytometer detection and select appropriate voltage settings). Based on the fluorescence intensity, the DNA replication activity during the S phase of the cell cycle can be determined. The fluorescent dye iF555 (Reagent B) in this kit has a spectral property of Ex/Em: 557 nm/570 nm (red); Hoechst 33342 staining solution has a spectral property of Ex/Em: 346 nm/460 nm (blue).

Notes:

1. For in vitro cultured cells, the specific concentration and incubation time of EdU may vary depending on the sample and research objectives, and adjustments can be made accordingly.
2. Some tissue cells have slow proliferation rates. To exclude factors such as poor modeling effects, it is recommended to select tissues with fast proliferation rates as reference samples (e.g., intestinal tissue).
3. If the background color is too dark, it may be due to inadequate washing during the experiment, prolonged fixation time of tissue samples, or residual fixation solution.
4. The Catalyst Additive (Reagent C) of EdU is prone to oxidation. Avoid prolonged exposure to air. It is recommended to store it in aliquots after preparing the aqueous solution. It has been tested that if the color of the Catalyst Additive changes slightly, the Click reaction catalytic system can still proceed normally. However, if it turns brown, it indicates that the component has become ineffective and should be discarded.
5. Please wear appropriate laboratory attire and disposable gloves when performing the procedures.

This product is intended for research use only and is not for clinical diagnostic purposes.

Version: V1.0-202101