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Cat. No.: ECK594-500 (for 50-500T)

Cat. No.: ECK594-2k (for 200-2000T)

Description

EdU Cell Proliferation Kit with Alexa Fluor 594 is a reagent based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analog, during DNA synthesis. Subsequent Click reaction allows EdU to be labeled with Alexa Fluor 594, enabling simple, rapid, and highly sensitive detection of cell proliferation.

This kit can detect individual proliferating cells in cell or tissue samples, as well as quantitatively analyze the overall cell proliferation status of cell or tissue samples. It is applicable to cultured cells or tissue samples and can also be used for tissue sections.

Following treatment with this kit, proliferating cells exhibit bright red fluorescence under a fluorescence microscope, suitable for detection using fluorescence microscopy, laser confocal microscopy, flow cytometry, or fluorescence enzyme immunoassay. However, flow cytometry or fluorescence enzyme immunoassay detection is only suitable for cell samples and not for tissue sections.

The detection of cell proliferation capacity is a fundamental method for evaluating cell activity, genotoxicity, and the effectiveness of anti-tumor drugs. The most accurate method recognized for detecting cell proliferation is to directly detect DNA synthesis within cells. Initially, the widely used method for detecting cell proliferation by monitoring DNA synthesis was the radioactive labeling nucleoside incorporation method, such as [3H]thymidine incorporation. However, this method was greatly limited due to radioactive contamination and the difficulty of achieving single-cell detection. It was gradually replaced by the BrdU (bromo-deoxyuridine) method based on antibody detection. However, the BrdU method is cumbersome, requiring the use of BrdU antibodies, and has many influencing factors with relatively poor stability. Additionally, interference may occur with other protein detection based on antibodies. The EdU method, based on EdU incorporation and subsequent Click reaction, does not require the use of antibodies, offering convenient operation and high detection sensitivity. It represents a new method that upgrades and replaces the BrdU method.

Methods such as MTT assay, WST-1 assay, CCK-8 assay, and CellTiter-Lumi™ chemiluminescence assay are all cell proliferation detection methods based on cell activity, capable of detecting the overall proliferation effect of cells but unable to detect individual proliferating cells. Although these methods do not detect DNA synthesis, they are widely used to replace [3H]thymidine incorporation. The CFDA SE method, based on cell fluorescence tracing principles, can detect individual proliferating cells. However, due to fluorescence attenuation with each proliferation cycle, it is difficult to distinguish cells with halved fluorescence under a fluorescence microscope, limiting its sensitivity, typically suitable only for flow cytometry detection. In scientific research, these methods based on cell activity or CFDA SE can serve as supplementary detection methods for the EdU method.

EdU (5-ethynyl-2'-deoxyuridine), also known as 5-ethynyl-2'-deoxyuridine, is a novel thymidine analog. EdU can replace thymidine and be incorporated into newly synthesized DNA during DNA synthesis. Moreover, the alkyne group on EdU can undergo a covalent reaction with fluorescently labeled small molecule azide probes (such as Azide Alexa Fluor 488, Azide Alexa Fluor 555, Azide Alexa Fluor 594, Azide Alexa Fluor 647, etc.) catalyzed by copper ions, rapidly forming stable triazole rings. This reaction, known as the Click reaction, allows newly synthesized DNA to be labeled with the corresponding fluorescent probes, enabling the detection of proliferating cells using appropriate fluorescence detection equipment.

Components

• EdU (10mM)
• Azide 594
• Click Reaction Buffer
• CuSO4
• Click Additive
• Hoechst 33342 (1000X)

Specification

The small package of the kit can detect 50 samples when used for cell cultures (6-well plate), with a reaction volume of 500 μl of Click reaction solution per sample. If used for detection in a 96-well plate, it can detect 500 samples, with a detection system volume of 50 μl of Click reaction solution per sample. For detection in 12-well, 24-well, 48-well, or 384-well plates, it can detect 125, 250, 350, and 1250 samples respectively, with recommended volumes of 200 μl, 100 μl, 70 μl, and 20 μl of Click reaction solution per sample.

For flow cytometry detection, the small package can detect 50 samples, with an ideal cell quantity of 10-100 million cells per sample, and a reaction volume of 500 μl of Click reaction solution per sample.

For detection in frozen or paraffin-embedded sections, the small package can detect 125-250 samples, with a reaction volume of 100-200 μl of Click reaction solution per sample.

The large package can detect four times the number of samples as the small package.

Features

• This kit offers simple reaction procedures and high detection sensitivity. Based on a straightforward and efficient Click reaction, it efficiently labels incorporated EdU without the need for DNA denaturation, requiring only a small amount of small molecule azide probes to effectively mark the EdU incorporation, enabling the detection of individual cell proliferation.
• The kit is convenient to use and exhibits good compatibility. With only common formaldehyde fixation and Triton X-100 permeabilization, the azide probes can effectively enter cells and undergo Click reaction, without affecting cell morphology, antibody-based immunofluorescence, immunohistochemistry, or DNA fluorescence staining (such as PI staining for cell cycle detection, DAPI or Hoechst dyes for nuclear staining). In contrast, the BrdU method requires denaturation of double-stranded DNA (such as acid denaturation, heat denaturation, or DNase digestion) to allow large BrdU antibodies to enter cells and bind to BrdU on DNA, which may affect cell morphology and subsequent immunofluorescence, immunohistochemistry, and DNA fluorescence staining.
• This kit offers rapid detection, making both qualitative and quantitative analyses highly convenient. Compared to the BrdU method, which typically requires at least 4 hours, our proprietary EdU method employed by this kit for detecting newly synthesized DNA takes only 1.5-2 hours, significantly reducing the time required. Additionally, the kit provides Hoechst 33342 for staining cell nuclei, facilitating the observation of all cell nuclei. Qualitative and quantitative analyses can be performed using fluorescence microscopy or flow cytometry.

Storage

Store at -20°C, valid for one year. Azide 594 and Hoechst 33342 should be stored protected from light.

Notes

• After preparing the Click Additive into a solution, please ensure appropriate aliquoting. If white precipitates appear after dissolution, invert the solution several times until fully dissolved before use. If the solution turns brown, it indicates that the active component of this ingredient has deteriorated, and the solution should be discarded.
• If hydroxyurea is needed as a control, it can be ordered from us. 
• If more EdU is required for animal experiments, it can also be ordered from us.
• Due to the copper ion catalysis required for the click reaction, please note the following compatibility issues and solutions: This product is fully compatible with organic dyes such as the Alexa Fluor® series and fluorescein (FITC), Allophycocyanin (APC), and APCE-tandem dyes. For Qdot® nanocrystal probes, Horseradish peroxidase (HRP), R-phycoerythrin (R-PE), and R-PE-tandem dyes such as Alexa Fluor® 680-R-PE, reactions and detection should be performed after the click reaction is completed. This product will affect the fluorescence of GFP, RFP, mCherry, and similar fluorescent proteins. For fluorescent proteins such as Green Fluorescent Protein (GFP), TC-FlAsH™, and TC-ReAsH™ reagents, reactions and detection should be performed before the click reaction. Since Phalloidin is not compatible with the click reaction, it is recommended to use Tubulin-Tracker Red for microtubule detection.
• This product is for scientific research by professionals only and should not be used for clinical diagnosis or treatment, food or drug purposes, or stored in ordinary residences.
• For your safety and health, wear laboratory clothing and disposable gloves during operation.

Only for research and not intended for treatment of humans or animals

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SBS Genetech is a long-term sponsor of Cold Spring Harbor Laboratory