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Cat. No.: ECK488-500 (for 50-500T)

Cat. No.: ECK488-2k (for 200-2000T)

Description

EdU Cell Proliferation Kit with Alexa Fluor 488 is a reagent kit designed for the simple, rapid, and highly sensitive detection of cell proliferation. It is based on the incorporation of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analogue, into DNA during the synthesis process. Subsequently, EdU is labeled with Alexa Fluor 488 through a click reaction, enabling the visualization of proliferating cells.

This kit can detect individual proliferating cells within cell or tissue samples, as well as quantitatively assess overall cell proliferation. It is applicable for cultured cell or tissue samples, as well as tissue sections. Proliferating cells treated with this kit exhibit bright green fluorescence under fluorescent microscopy, suitable for detection using various instruments such as fluorescent microscopes, laser scanning confocal microscopes, flow cytometers, or fluorescent plate readers. However, flow cytometry or fluorescent plate reader detection is only applicable to cell samples and not tissue sections.

Detection of cell proliferation capacity is fundamental for assessing cell viability, genotoxicity, and the efficacy of anti-tumor drugs. The most accurate method for detecting cell proliferation is by directly measuring DNA synthesis within cells. Initially, the widely used method for detecting cell proliferation by measuring DNA synthesis was the radioactive nucleoside incorporation method, such as [3H]thymidine incorporation. However, this method was greatly limited due to radioactive contamination and difficulties in single-cell detection, leading to its gradual replacement by the BrdU (bromo-deoxyuridine) method based on antibody detection. The BrdU method is more complex, requires the use of BrdU antibodies, is affected by multiple factors, and has poorer stability. Additionally, interference may occur with other antibody-based detections. The EdU method, based on EdU incorporation and subsequent click reaction, does not require the use of antibodies, is easy to perform, and offers high detection sensitivity. It represents a new method that upgrades and replaces the BrdU method, gradually becoming its successor.

Methods such as the MTT assay, WST-1 assay, CCK-8 assay, and chemiluminescence assay are all based on cell viability and proliferation detection but cannot detect individual proliferating cells. These methods, although not based on DNA synthesis detection, are widely used as alternatives to [3H]thymidine incorporation. The CFDA SE method, based on fluorescent tracking of cells, can detect individual proliferating cells, but its sensitivity is not very high due to fluorescence diminishing by half with each proliferation cycle. It is usually only applicable for flow cytometry detection. In scientific research, these methods based on cell viability or CFDA SE can serve as complementary detection methods to the EdU method.

EdU (5-ethynyl-2'-deoxyuridine), also known as 5-ethynyl-2'-deoxyuridine, is a novel thymidine analogue that can replace thymidine during DNA synthesis. On the other hand, the ethynyl group on EdU can undergo a covalent reaction with fluorescent-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 a monovalent copper ion, forming a stable triazole ring. This reaction, called the click reaction, is very rapid. Newly synthesized DNA is labeled with the corresponding fluorescent probe through the click reaction, enabling the detection of proliferating cells using appropriate fluorescence detection equipment.

Components

• EdU (10mM)
• Azide 488
• Click Reaction Buffer
• CuSO4
• Click Additive
• Hoechst 33342 (1000X)

Specification

For the small package of this kit, if used for the detection of cells cultured in a 6-well plate, it can test 50 samples, with each sample having a reaction system of 500μl of Click reaction solution. If used for detection in a 96-well plate, it can test 500 samples, with each sample having a detection system of 50μl of Click reaction solution. For detection in 12-well, 24-well, 48-well, or 384-well plates, it can test 125, 250, 350, and 1250 samples respectively. The recommended amounts of Click reaction solution for each sample are 200μl, 100μl, 70μl, and 20μl respectively.

For flow cytometry detection using the small package, it can test 50 samples, with each cell sample containing 10-100 thousand cells, and each sample having a reaction system of 500μl of Click reaction solution.

If used for frozen or paraffin-embedded section detection, the small package can test 125-250 samples, with each sample having a reaction system of 100-200μl of Click reaction solution.

The large package can test four times the number of samples compared to the small package.

Features

• This kit offers simple reactions and high detection sensitivity. Based on the straightforward and efficient click reaction, it requires no DNA denaturation. Only a small amount of azide probes is needed to effectively label the incorporated EdU, allowing for the detection of individual cell proliferation.
• The kit is convenient to use and compatible with various procedures. It only requires common polyformaldehyde fixation and Triton X-100 permeabilization, enabling the azide probes to efficiently enter cells and undergo click reactions without affecting cell morphology. This process does not interfere with antibody-based immunofluorescence and immunohistochemistry assays, nor does it affect DNA fluorescence staining (such as PI staining for cell cycle analysis, or 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. This denaturation process may affect cell morphology and subsequent immunofluorescence, immunohistochemistry assays, and DNA fluorescence staining.
• This kit enables rapid detection, both qualitative and quantitative, in a convenient manner. Compared to the BrdU method, which typically takes at least 4 hours, our proprietary EdU method utilized by this kit for detecting newly synthesized DNA only requires 1.5-2 hours, significantly reducing the processing time. Additionally, the kit provides Hoechst 33342 for staining cell nuclei, facilitating 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 488 and Hoechst 33342 should be stored away from light.

Notes

• After preparing the Click Additive into a solution, please ensure proper aliquoting. If white precipitates appear after dissolution, invert the solution several times until completely dissolved before use. If the solution turns brown, it indicates that the effective component of that component has deteriorated, so please discard.
• 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 necessity of copper ion catalysis for the click reaction, please be aware of the following compatibility issues and solutions. This product is fully compatible with organic dyes such as the Alexa Fluor® series and common dyes like fluorescein (FITC), Allophycocyanin (APC), and APC-E tandems; for Qdot® nanocrystal probes, Horseradish peroxidase (HRP), R-phycoerythrin (R-PE), and R-PE tandems such as Alexa Fluor® 680-R-PE, reaction and detection should be performed after the click reaction is completed; this product will affect the fluorescence of GFP, RFP, mCherry, etc., so for fluorescent proteins like Green Fluorescent Protein (GFP), TC-FlAsH™, and TC-ReAsH™ reagents, reaction and detection should be performed before the click reaction. Due to the incompatibility of Phalloidin 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 is not intended for clinical diagnosis or treatment, food, or drugs, and should not be stored in residential homes.
• For your safety and health, please wear lab coats 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