EdU Cell Proliferation Detection Kit (AF488)

Cat. No.: E1373491

QUALITÀ & PUREZZA Bioactive, Biological Stain, for Fluorescence analysis, for Microscopy

Panoramica

Product Introduction

Cell proliferation assays are widely used in the evaluation of cell viability, genotoxicity, and the efficacy of antitumor drugs. Direct detection of DNA synthesis in cells is considered the most accurate method for assessing cell proliferation. EdU (5-ethynyl-2′-deoxyuridine) is a novel thymidine (thymine deoxyribonucleoside) analogue. During DNA synthesis, EdU can be incorporated into newly synthesized DNA in place of thymidine. The ethynyl group on EdU can undergo a covalent reaction with fluorescently labeled small-molecule azide probes (such as Azide AF 488, Azide AF 555, Azide AF 594, Azide AF 647, etc.) via Cu(I)-catalyzed click chemistry, forming a stable triazole ring. This reaction is highly efficient and is referred to as the Click reaction. Through this process, newly synthesized DNA is labeled with the corresponding fluorescent probes, enabling the detection of proliferating cells using appropriate fluorescence detection equipment. 

Usage Protocol

1. Preparation

1) Preparation of Click Additive Solution: 

For a 50-test kit: Add 12.5 mL of pre-chilled deionized water to the tube. Mix thoroughly until completely dissolved to obtain the Click Additive Solution. For a 100-test kit: Add 25 mL of pre-chilled deionized water to the tube. Mix thoroughly until completely dissolved to obtain the Click Additive Solution. For a 200-test kit: Add 50 mL of pre-chilled deionized water to the tube. Mix thoroughly until completely dissolved to obtain the Click Additive Solution. After preparation, aliquot the solution as needed and store at -20°C. If a white precipitate forms after dissolution, invert the tube repeatedly until it is fully dissolved before use. If the solution turns brown, it indicates degradation of the active component; discard it.

2) Upon initial dissolution of the Click Reaction Buffer, aliquot it according to the number of samples per experiment and store at -20°C.

2. EdU Labeling of Cells

It is recommended to use a final EdU concentration of 10 μM (1×). A 1:500 dilution of EdU (10 mM) in cell culture medium yields a 2× EdU working solution (20 μM). Mix an equal volume of pre-warmed (37°C) 2× EdU working solution (20 μM) with the cell suspension to achieve a final 1× EdU concentration. Incubate in a 37°C, 5% CO₂ incubator. Factors such as cell culture medium, cell density, cell type, and other experimental conditions may affect labeling efficiency. Therefore, the optimal EdU concentration and labeling duration must be empirically determined based on the cell type under investigation.

3. Fixation and Permeabilization

1) Harvest cells and centrifuge at 300 ×g for 5 min. Wash cells twice with  PBS containing 2% FBS.

2) Fix cells with 4% paraformaldehyde solution. Mix thoroughly and incubate for 15 min at room temperature protected from light.

3) Collect cells and centrifuge at 300 × g for 5 min. Wash cells twice.

4) Resuspend cells in PBS containing 0.1% Triton X-100. Mix well and incubate for 15 min at room temperature.

5) Centrifuge at 300 × g for 5 min and wash cells twice.

4. Fluorescent Labeling

1) This protocol is based on a 500 μL reaction system per 2 × 10⁶ cells. The volume of the Click reaction mixture can be adjusted according to the experimental sample size.

2) Centrifuge the cells at 300 ×g for 5 minutes. Add 500 μL of Click reaction mixture per sample, mix gently, and incubate for 30 minutes at room temperature protected from light.

3) After the reaction, wash the cells twice with PBS containing 2% FBS.

4) Dilute the DAPI Staining Solution (1000×) to 1× using PBS containing 2% FBS. Add 250 μL of the diluted DAPI solution to each sample and incubate for 5 minutes at room temperature.

5) Add an additional 250 μL of PBS containing 2% FBS, mix gently, and proceed to detection using an appropriate flow cytometry instrument

Precautions

1. Strictly adhere to the component order and volumes specified in the table above when preparing the Click reaction mixture, as deviations may affect subsequent experimental results.

2. The Click reaction mixture must be used within 15 minutes of preparation.

3. To avoid fluorescence quenching, perform detection as soon as possible after sample preparation.

Specifiche

Sinonimi

VF 488 Click-iT EdU Universal Cell Proliferation Detection Kit

Specifiche e purezza

Bioactive, Biological Stain, for fluorescence analysis, for microscopy

Grado

Bioactive, Biological Stain, per l'analisi a fluorescenza, per la microscopia

Nota

This product can replace A598377.

Applicazione

Cell proliferation, Flow Cytometry

Sensibilità

light-sensitive

Stoccaggio e spedizione

Condizioni di conservazione di stoccaggio

Protected from light,Store at -20°C

Spedito in

Ice chest + Ice pads

Stabilità e conservazione

Store at -20℃ long term (12 months)

Contents & Storage

E1373491	Component	50 T	100 T	200 T	Storage conditions	Quantity Per Test
E1373491A	EdU(10 mM)	100 μL	200 μL	400 μL	-20℃.Store in the dark.	2 μL  per 1.0-2.0 × 10⁶ cells
E1373491B	AF488 azide	100 μL	200 μL	400 μL	-20℃.Store in the dark.	2 μL  per 1.0-2.0 × 10⁶ cells
E1373491C	Click Reaction Buffer	13 mL	26 mL	52 mL	-20℃.Store in the dark.	243 μL  per 1.0-2.0 × 10⁶ cells
E1373491D	CuSO₄	250 μL	500 μL	1000 μL	-20℃.	5 μL  per 1.0-2.0 × 10⁶ cells
E1373491E	Click Additive	248 mg	496 mg	992 mg	-20℃.Store in the dark.	250 μL  per 1.0-2.0 × 10⁶ cells
E1373491F	DAPI Staining Solution（1000×）	25 μL	50 μL	100 μL	-20℃.Store in the dark.	0.5 μL  per 1.0-2.0 × 10⁶ cells

Immagini

Flow Cytometry: EdU Cell Proliferation Detection Kit (AF488)(E1373491)

Flow Cytometry: EdU Cell Proliferation Detection Kit (AF488)(E1373491)

Jurkat (human T-cell leukemia) cells were treated with 10 µM EdU (E131265) for 2 hours and detected according to the recommended staining protocol. The incorporated EdU was then conjugated to AF 488 azide via a click reaction and analyzed by flow cytometry using 488 nm (for AF 488) and 405 nm (for DAPI) excitation. (A) Histogram of EdU-AF 488 signal, distinguishing EdU-positive S-phase cells from cells in other cell cycle phases. (B) DNA content histogram (DAPI) resolving the G0/G1, S, and G2/M populations. (C) Dot plot of EdU incorporation versus DNA content, allowing for direct quantification of S-phase cells.