Shop All Cell Proliferation Assay Kits

CellTrace™ Violet Cell Proliferation Kit, for flow cytometry (Invitrogen™)

CellTrace™ Violet Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry.

• Superior performance—bright, single-peak staining enables visualization of multiple generations
• Long-term signal stability—well-retained in cells for several days post stain
• Versatile—multiple colors available to easily combine with antibodies or markers of cell function, such as GFP
• Simple, robust staining protocol

View a selection guide for all CellTrace™ Cell Proliferation Kits for flow cytometry.

Superior fluorescent staining
Successful proliferation analysis by dye dilution (see figure below) requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace™ Violet dye enables the visualization of ten or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. Consistent, homogeneous staining results in very little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace™ Violet stain easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye provides a consistent signal, even after several days in a cell culture environment. CellTrace™ Violet dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells.

Easy multiplexing with other fluorophores
The violet excitation and narrow emission of CellTrace™ Violet dye make it ideal for multiplexing due to the limited spectral overlap with other common dyes (Alexa Fluor™ 488, FITC, and RPE) and fluorescent proteins (Green Fluorescent Protein (GFP) and mCherry) (see Fluorescence Spectra for CellTrace™ Violet stain below).

Simple, robust staining protocol
The CellTrace™ Violet Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

CellTrace™ Far Red Cell Proliferation Kit, for flow cytometry (Invitrogen™)

CellTrace™ Far Red Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry.

• Superior performance—bright, single-peak staining enables visualization of multiple generations
• Long-term signal stability—well-retained in cells for several days post stain
• Versatile—multiple colors available to easily combine with antibodies or markers of cell function, such as GFP
• Simple, robust staining protocol

View a selection guide for all CellTrace™ Cell Proliferation Kits for flow cytometry.

Superior fluorescent staining
Successful proliferation analysis by dye dilution requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace™ Far Red dye enables the visualization of six or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. This kit enables consistent, homogeneous staining results with very little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace™ Far Red stain easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye offers a consistent signal, even after several days in a cell culture environment. CellTrace™ Far Red dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells.

Easy multiplexing with other fluorophores
The red excitation at 630 nm and emission at 661 nm of CellTrace™ Far Red dye make it ideal for multiplexing due to the limited spectral overlap with other common dyes (Alexa Fluor™ 488, FITC, and RPE) and fluorescent proteins (Green Fluorescent Protein (GFP) and mCherry).

Simple, robust staining protocol
The CellTrace™ Far Red Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

CellTrace™ Yellow Cell Proliferation Kit, for flow cytometry (Invitrogen™)

CellTrace™ Yellow Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry. This reagent is specifically designed for excitation by either a 532-nm or 561-nm laser, and it can be easily multiplexed with reagents and antibodies excited by other commonly used lasers.

Superior performance—bright, single-peak staining enables visualization of multiple generations
Long-term signal stability—well retained in cells for several days post stain
Versatile—multiple colors available to easily combine with antibodies or markers of cell function
Simple—robust staining protocol

Additional information is available about other CellTrace™ Reagents to meet your multiplexing needs.

Superior fluorescent staining
Successful proliferation analysis by dye dilution requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace Yellow dye enables the visualization of six or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. This kit enables consistent, homogeneous staining results with little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace Yellow stain easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye offers a consistent signal, even after several days in a cell culture environment. CellTrace Yellow dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells.

Easy multiplexing with other fluorophores
The yellow excitation at 546 nm and emission at 579 nm of CellTrace Yellow dye make it ideal for multiplexing due to the limited spectral overlap with other common dyes (e.g., Alexa Fluor™488 and FITC) and green fluorescent protein (GFP) .

Use the SpectraViewer to help you plan your experiments.

Simple, robust staining protocol
The CellTrace Yellow Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

Related links
General information about Thermo Fisher Scientific flow cytometry products and support is also available.

CellTrace™ Blue Cell Proliferation Kit, for flow cytometry (Invitrogen™)

The CellTrace Blue Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry. The CellTrace Blue reagent is specifically designed for excitation by a UV laser (at 355 nm or 375 nm), and it can be easily multiplexed with reagents and antibodies excited by other commonly used lasers.

• Superior performance—bright, single-peak staining enables visualization of multiple generations
• Long-term signal stability—well-retained in cells for several days post stain
• Versatile—multiple colors available to easily combine with antibodies or markers of cell function, such as GFP
• Simple, robust staining protocol

View a selection guide for all CellTrace Cell Proliferation Kits for flow cytometry.

Superior fluorescent staining
Successful proliferation analysis by dye dilution (see figure below) requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace Blue dye enables the visualization of five or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. Consistent, homogeneous staining results in very little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.The recommended working concentration for peripheral blood mononuclear cells is 5–10 µM. However, as with all reagents used in flow cytometry, to achieve optimal signal, reagent titration is highly recommended.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace Blue stain easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye provides a consistent signal, even after several days in a cell culture environment. CellTrace Blue dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells.

Easy multiplexing with other fluorophores
The UV excitation and narrow emission of CellTrace Blue dye make it ideal for multiplexing as it lies outside the spectral emission profiles of most other dyes and fluorescent proteins.

Simple, robust staining protocol
The CellTrace Blue Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

CyQUANT™ NF Cell Proliferation Assay (Invitrogen™)

The CyQUANT® NF Cell Proliferation Assay provides a fast and sensitive method for counting cells in a population and measuring proliferation in microplate format.

Features of the CyQUANT® NF Cell Proliferation Assay:

• More sensitive than MTT or AlamarBlue® assays
• Linear detection range from 100 to 20,000 cells per well (96-well microplate)
• Assays can be completed in one hour

A Rapid and simple method for measuring cell proliferation
The CyQUANT® NF Cell Proliferation Assay does not require cell lysis, long incubations, radioactivity, or removal of stain from cells. The CyQUANT® NF assay eliminates the freeze-thaw cell lysis step of the original CyQUANT® cell proliferation assay by using a cell-permeant DNA-binding dye in combination with a plasma membrane permeabilization reagent. The CyQUANT® NF Cell Proliferation Assay can be used in either 96-well or 384-well microplate formats, and is available in two configurations: a 200 assay kit (C35007) for smaller sample sizes, and a 1000 assay kit (C35006) for high-throughput applications.

AlamarBlue® is a registered trademark of TREK Diagnostic Systems.

eBioscience™ BrdU Staining Kit for Flow Cytometry FITC (Invitrogen™)

The eBioscience BrdU Staining Kit for Flow Cytometry contains the necessary reagents and buffers for identifying and examining proliferating cells of mammalian species by flow cytometric analysis. Cycling cells are incubated with 5-bromo-2’-deoxyuridine (BrdU), a synthetic analog of thymidine which incorporates into newly synthesized genomic DNA during the S-phase of mitosis. Following DNA denaturation, the cells are stained for BrdU incorporation along with any other cell surface and/or intracellular targets of interest.

This kit is optimized to achieve brighter staining compared to traditional protocols.

Reactivity/Species
Bovine, Canine, Cow, Human, Monkey, Mouse, Non-human primate, Rat, Rhesus

Conjugate
FITC

Laser
Blue Laser

Emit
520 nm

Excite
488 nm

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

CellTrace™ CFSE Cell Proliferation Kit, for flow cytometry (Invitrogen™)

CellTrace™ CFSE Cell Proliferation Kit is used for in vitro and in vivo labeling of cells to trace multiple generations using dye dilution by flow cytometry.

• Superior performance—bright, single-peak staining enables visualization of multiple generations
• Long-term signal stability—well-retained in cells for several days post stain
• Non-toxic—staining does not adversely effect cell health
• Simple, robust staining protocol

View a selection guide for all CellTrace™ Cell Proliferation Kits for flow cytometry.

Superior fluorescent staining
Successful proliferation analysis by dye dilution (see figure below) requires an extremely bright dye to distinguish fluorescently labeled cells from auto-fluorescence after several cell divisions. The intense fluorescent staining provided by CellTrace™ CFSE dye enables the visualization of eight or more generations of proliferating cells before the signal is overwhelmed by intrinsic cellular auto-fluorescence. Consistent, homogeneous staining results in very little fluorescence variation between cells in a population, so distinct generations can be seen without any requirement for complex modeling software.

Long-term signal retention
Unlike stains that label the lipid membrane of cells, CellTrace™ CFSE dye easily crosses the plasma membrane and covalently binds inside cells where the stable, well-retained fluorescent dye is designed to provide a consistent signal, even after several days in a cell culture environment.

Non-toxic
CellTrace™ CFSE dye binds covalently to all free amines on the surface and inside of cells and shows little cytotoxicity, with minimal observed effect on the proliferative ability or biology of cells. Researchers have used CFSE SE labeling to show that transplantable hematopoietic cells proliferate in vitro in response to stimulation by a growth factor cocktail.

Simple, robust staining protocol
The CellTrace™ CFSE Cell Proliferation Kit contains convenient single-use vials of dry dye to permit small-scale experiments without preparing excess quantities of dye. A stock solution is prepared by dissolving the contents of a vial in anhydrous DMSO prior to use. To stain 1 mL of cells in protein-free medium, 1 µL of this stock solution is typically used. Cells should be stained for 20 minutes at room temperature with gentle agitation. A brief wash with complete medium will then quench any dye remaining in solution.

Click-iT™ EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit (Invitrogen™)

The Click-iT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells as compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the 488 nm laser of the flow cytometer.

Accurate—superior results compared to BrdU assays
Fast—results in as little as 90 minutes
Economical—more assays per kit

View selection guide for all Click-iT™ EdU and Click-iT™ Plus EdU assays for flow cytometry.

An Advanced Method Giving You Results Superior to BrdU
The most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides, i.e. 3H-thymidine. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT® EdU Flow Cytometry Assay Kits are novel alternatives to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog which is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry; a copper catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to Alexa Fluor ® 488, Alexa Fluor® 647, or Pacific BlueTM dyes. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population (Fig 1).

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies
The advantages of Click-iT® EdU labeling are readily evident while performing the assay (Figure 2). The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it may be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use EdU cell proliferation kit is compatible with cell cycle dyes. This EdU assay kit can also be multiplexed with antibodies against surface and intracellular markers.

Quick and Simple Protocol
The Click-iT® EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling, but EdU is compatible with other
fixation/permeabilization agents including saponin and methanol. In just five steps you’ll be ready to analyze your cell proliferation data:

• Treat cells with EdU
• Fix and permeabilize cells
• Detect S-phase cells with Click-iT® detection cocktail for 30 min
• Wash once
• Analyze

Get Accurate Results Economically
By increasing the number of assays per kit, the Click-IT® EdU Alexa Fluor® 488 Flow Cytometry Assay Kit is less expensive than the traditional BrdU assays making them ideal for large experiments.

For research use only. Not intended for any human or animal therapeutic of diagnostic use.

Click-iT™ EdU Imaging Kit with Alexa Fluor™ 488, 594, and 647 Azides (Invitrogen™)

The Click-iT® EdU Imaging Kit is a trial version of our superior alternative to traditional proliferation assays optimized for fluorescence microscopy applications. In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor® dye in a fast, highly-specific click reaction. This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol. This trial-size kit provides sufficient EdU to label up to 6–30 coverslips and enough Alexa Fluor® 488, 594, and 647 azide to detect new DNA synthesis on 2–10 coverslips with each fluorophore.
• Simple—works the first time, every time, in less time
• Efficient—no denaturation steps or harsh treatment required
• Content-rich results—better preservation of cell morphology, antigen structure, and DNA integrity
• Consistent—not dependent on variable antibody lots for detection

The most accurate proliferation-detection methods are based on the incorporation and measurement of nucleoside analogues in newly synthesized DNA, with bromodeoxyuridine (BrdU) a commonly used analogue. BrdU-labeled DNA is quantitated using anti-BrdU antibodies following DNA denaturation by harsh methods (HCl, heat, or enzymes) to expose the BrdU molecules. This step is time consuming and difficult to perform consistently. The harsh treatment can also adversely effect sample integrity and quality, which makes co-staining with other antibodies challenging.

Superior Proliferation Methodology
The Click-iT® EdU Imaging Kit provides a superior alternative to BrdU assays for measuring cell proliferation. EdU (5-ethynyl-2'-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. With Click-iT® EdU, mild fixation and detergent permeabilization is sufficient for the small molecule-based Click-iT® EdU detection reagent to gain access to the DNA. As a consequence, the Click-iT® EdU Imaging Kit is not only easy to use, but more accurate and compatible with cell cycle analysis and other intracellular or extracellular targets for truly content-rich results.

This kit is optimized for fluorescence microscopy applications; visit the Click-iT® technology area of our website for kits designed for high-content imaging, fluorescence microplate, or flow cytometry platforms.

Learn more about Click-iT® technology

Notes:
The Click-iT® assay can be used on cells in culture or in vivo following EdU administration by feeding or injection methods.
The Click-iT® assay can be used with BrdU in dual pulse experiments by using the anti-BrdU (clone MoBu-1) antibody, which does not cross react with EdU.
The Click-iT® technology is compatible with immunohistochemical, immunocytochemical, and fluorescent dyes that are fixation tolerant or designed for fixed cell labeling.

eBioscience™ BrdU Staining Kit for Flow Cytometry PerCP-eFluor™ 710 (Invitrogen™)

The eBioscience BrdU Staining Kit for Flow Cytometry contains the necessary reagents and buffers for identifying and examining proliferating cells of mammalian species by flow cytometric analysis. Cycling cells are incubated with 5-bromo-2’-deoxyuridine (BrdU), a synthetic analog of thymidine which incorporates into newly synthesized genomic DNA during the S-phase of mitosis. Following DNA denaturation, the cells are stained for BrdU incorporation along with any other cell surface and/or intracellular targets of interest.

This kit is optimized to achieve brighter staining compared to traditional protocols.

Reactivity/Species
Bovine, Canine, Cow, Human, Monkey, Mouse, Non-human primate, Rat, Rhesus

Conjugate
PerCP-eFluor 710

Laser
Blue Laser

Emit
710 nm

Excite
488 nm

Reported Application
Intracellular Staining Followed by Flow Cytometric Analysis

Click-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit (Invitrogen™)

The Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the 488 nm laser of the flow cytometer. The Click-iT® Plus formulation is compatible with standard fluorophores, including R-PE and R-PE tandems, as well as fluorescent proteins.

• Multiplexable—compatible with R-PE (and tandems) and fluorescent proteins
• Accurate—superior results compared to BrdU assays
• Fast—results in as little as 60 minutes

View selection guide for all Click-iT™ EdU and Click-iT™ Plus EdU assays for flow cytometry.

Multiplexable
The Click-iT® Plus formulation provides increased multiplexibility compared to the original Click-iT® EdU Flow Cytometry assays. Click-iT® Plus EdU assays can be used in conjunction with R-PE and R-PE tandems, as well as fluorescent proteins such as GFP and mCherry, without loss of the accuracy or speed of the original Click-iT® EdU assay.

An Advanced Method Giving You Results Superior to BrdU
The most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT® Plus EdU Flow Cytometry assay is a novel alternative to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog that is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry, which is a copper-catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to the Alexa Fluor® dye. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population.

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies
The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® Plus detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it can be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution, as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use Click-iT® Plus EdU assay is compatible with cell cycle dyes. The Click-iT® Plus EdU assay can also be multiplexed with antibodies against surface and intracellular markers, as well as conjugates labeled with standard fluorophores including R-PE, R-PE tandems, and fluorescent proteins (GFP and mCherry).

Quick and Simple Protocol
The Click-iT® Plus EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling. However, EdU is compatible with other fixation/permeabilization agents including saponin and methanol. In just five steps you’ll be ready to analyze your cell proliferation data:

1. Treat cells with EdU.
2. Fix and permeabilize cells.
3. Detect S-phase cells with Click-iT® Plus detection cocktail for 30 min.
4. Wash once.
5. Analyze.

Results can be seen in as little as 60 minutes in some circumstances, but we recommend 90 minutes for all applications.

Click-iT™ Plus EdU Alexa Fluor™ 350 Flow Cytometry Assay Kit (Invitrogen™)

The Click-iT Plus EdU Alexa Fluor 350 Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the UV laser of the flow cytometer. The Click-iT Plus formulation is compatible with standard fluorophores, including R-PE and R-PE tandems, as well as fluorescent proteins.

• Multiplexable—compatible with R-PE (and tandems) and fluorescent proteins
• Accurate—superior results compared to BrdU assays
• Fast—results in as little as 60 minutes

View selection guide for all Click-iT EdU and Click-iT Plus EdU assays for flow cytometry.

Multiplexable
Click-iT Plus Alexa Fluor 350 EdU assays can be used in conjunction with R-PE and R-PE tandems, as well as fluorescent proteins such as GFP and mCherry. The Alexa Fluor 350 labeling reagent is excited readily at 350 nm with a UV laser and emits at 438 nm.

An Advanced Method Giving You Results Superior to BrdU
The most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT Plus EdU Flow Cytometry assay is a novel alternative to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog that is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry, which is a copper-catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to the Alexa Fluor dye. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population.

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies
The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT Plus detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it can be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution, as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use Click-iT Plus EdU assay is compatible with cell cycle dyes. The Click-iT Plus EdU assay can also be multiplexed with antibodies against surface and intracellular markers, as well as conjugates labeled with standard fluorophores including R-PE, R-PE tandems, and fluorescent proteins (GFP and mCherry).

Quick and Simple Protocol
The Click-iT Plus EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling. However, EdU is compatible with other fixation/permeabilization agents including saponin and methanol. In just five steps you’ll be ready to analyze your cell proliferation data:

1. Treat cells with EdU.
2. Fix and permeabilize cells.
3. Detect S-phase cells with Click-iT Plus detection cocktail for 30 min.
4. Wash once.
5. Analyze.

Results can be seen in as little as 60 minutes in some circumstances, but we recommend 90 minutes for all applications.

Click-iT™ EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit (Invitrogen™)

The Click-iT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells as compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the 633/5 nm laser of the flow cytometer.

Accurate--Superior Results compared to BrdU Assays
Fast--Results in as little as 90 minutes
Economical--More assays per kit

View selection guide for all Click-iT™ EdU and Click-iT™ Plus EdU assays for flow cytometry.

An Advanced Method Giving You Results Superior to BrdU
The most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides, i.e., 3H-thymidine. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT® EdU Flow Cytometry Assay Kits are novel alternatives to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog which is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry: a copper catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to Alexa Fluor ® 488, Alexa Fluor® 647, or Pacific BlueTM dyes. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population (Fig 1).

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies
The advantages of Click-iT® EdU labeling are readily evident while performing the assay (Fig 2). The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it may be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use EdU cell proliferation kit is compatible with cell cycle dyes. This EdU assay kit can also be multiplexed with antibodies against surface and intracellular markers.

Quick and Simple Protocol
The Click-iT® EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling, but EdU is compatible with other
fixation/permeabilization agents including saponin and methanol. In just five steps you’ll be ready to analyze your cell proliferation data:

• Treat cells with EdUM
• Fix and permeabilize cells
• Detect S-phase cells with Click-iT® detection cocktail for 30 min
• Wash once
• Analyze

Get Accurate Results Economically
By increasing the number of assays per kit, the Click-IT® EdU Alexa Fluor® 647 Flow Cytometry Assay Kit is less expensive than the traditional BrdU assays making them ideal for large experiments.

For research use only. Not intended for any human or animal therapeutic of diagnostic use.

Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit (Invitrogen™)

The Click-iT® EdU Pacific Blue™ Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells as compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the 405 nm laser of the flow cytometer.

Accurate—superior results compared to BrdU assays
Fast—results in as little as 90 minutes
Economical—more assays per kit

View selection guide for all Click-iT™ EdU and Click-iT™ Plus EdU assays for flow cytometry.

An Advanced Method Giving You Results Superior to BrdU
The most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides, i.e., 3H-thymidine. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT® EdU Flow Cytometry Assay Kits are novel alternatives to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog which is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry: a copper catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to Alexa Fluor ® 488, Alexa Fluor® 647, or Pacific Blue™ dyes. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population (Fig 1).

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies
The advantages of Click-iT® EdU labeling are readily evident while performing the assay (Fig 2). The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it may be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use EdU cell proliferation kit is compatible with cell cycle dyes. This EdU assay kit can also be multiplexed with antibodies against surface and intracellular markers.

Quick and Simple Protocol
The Click-iT® EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling, but EdU is compatible with other fixation/permeabilization agents including saponin and methanol. In just five steps you’ll be ready to analyze your cell proliferation data:

• Treat cells with EdUM
• Fix and permeabilize cells
• Detect S-phase cells with Click-iT® detection cocktail for 30 min
• Wash once
• Analyze

Get Accurate Results Economically
By increasing the number of assays per kit, the Click-IT® EdU Pacific Blue™ Flow Cytometry Assay Kit is less expensive than the traditional BrdU assays making them ideal for large experiments.

For Research Use Only. Not for use in diagnostic procedures.

Click-iT™ EdU Microplate Assay (Invitrogen™)

The Click-iT® EdU Microplate Assay is a superior alternative to traditional cell proliferation assays that is optimized for microplate-based fluorescence analysis. This assay provides a simple and rapid workflow with reduced wash steps compared to traditional BrdU colorimetric or fluorescent proliferation assays. In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with bright, photostable Amplex® UltraRed dye in a fast, highly-specific click reaction. This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol.

• Simple—works the first time, every time, in less time
• Efficient—no denaturation steps or harsh treatment required
• Sensitive—outstanding sensitivity and dynamic range
• Versatile—fluorescence or absorbance readouts through use of Amplex® UltraRed reagent

The most accurate proliferation-detection methods are based on the incorporation and measurement of nucleoside analogues in newly synthesized DNA, with bromodeoxyuridine (BrdU) a commonly used analogue. BrdU-labeled DNA is quantitated using anti-BrdU antibodies following DNA denaturation by harsh methods (HCl, heat, or enzymes) to expose the BrdU molecules. This step is time consuming and difficult to perform consistently.

Superior Proliferation Methodology
The Click-iT® EdU Microplate Assay provides a superior alternative to BrdU assays for measuring cell proliferation. EdU (5-ethynyl-2'-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. With Click-iT® EdU, mild fixation and detergent permeabilization is sufficient for the small molecule-based Click-iT® EdU detection reagent to gain access to the DNA. The signal amplification steps include incubation with an anti-Oregon Green® antibody conjugated to horseradish peroxidase (HRP), which then reacts with Amplex® UltraRed substrate in a 1:1 stoichiometry and produces a bright red, fluorescent product (excitation/emission maxima ~568/585 nm).

With a high extinction coefficient, good quantum efficiency, and resistance to auto-oxidation, Amplex® UltraRed reagent delivers higher sensitivity and a broader assay range than other fluorogenic or colorimetric peroxidase substrates. Amplex® UltraRed reagent also offers versatility through detection by either fluorescence or absorbance measurement.

This kit also includes Amplex® UltraRed stop reagent that enables the peroxidase reaction to be terminated at a user-determined time point and stabilizes the signal for up to 24 hours. As a consequence, the Click-iT® EdU Microplate Assay is not only easy to use, but more accurate and compatible with cell cycle analysis and other intracellular or extracellular targets for truly content-rich results.

The Click-iT® EdU Microplate Assay is optimized for microplate-based fluorescence analysis; visit the Click-iT® technology area of our website for kits designed for fluorescence microscopy, high-content imaging, or flow cytometry platforms.

Learn more about Click-iT® technology

Notes:
The Click-iT® assay can be used on cells in culture or in vivo following administration of EdU by feeding or injection methods.
The Click-iT® assay can be used with BrdU in dual pulse experiments by using the anti-BrdU (clone MoBu-1) antibody, which does not cross react with EdU.
The Click-iT® technology is compatible with immunohistochemical, immunocytochemical, and fluorescent dyes that are fixation tolerant or designed for fixed-cell labeling.