Cell Proliferation for Imaging

Cell proliferation analyses are crucial for cell growth and differentiation studies, and are often used to evaluate both compound toxicity and inhibition of tumor cell growth during drug development. Proliferation measurements are typically made based on average DNA content or on cellular metabolism. Assays can report either total cell numbers or live cells, or measure DNA synthesis in single cells.

Our broad range of Invitrogen cell analysis assays and fluorescent labels can be used individually or together to carry out complex biological investigations, including studies of cell proliferation, cytotoxicity, or drug efficacy, using imaging, microplate, or flow cytometry assay platforms.

Select products for cell proliferation

New DNA synthesis—imaging/high-content analysis

Measuring the synthesis of new DNA is a precise way to assay cell proliferation in individual cells or in cell populations. Click-iT EdU technology measures the rate of new DNA synthesis based on incorporation of the nucleoside analog EdU into DNA. Detection is achieved through a copper-catalyzed "click" reaction that is complete typically within 30 minutes.

Click-iT EdU technology uses mild reaction conditions, and ultrabright Alexa Fluor "click" labels offer a range of fluorescence options, making these DNA synthesis assay reagents multiplex-compatible for content-rich and context-rich results. Assay kits are available for cultured cells or tissue sections analyzed using fluorescence microscopy, for HCS in microplates, or for microplate assays of cell lysates.

Microscopic image of cell proliferation as indicated by Click-iT Plus EdU

Multicolor imaging of HeLa cells with Click-iT Plus EdU Alexa Fluor 647 Imaging Kit.

New DNA synthesis—HTS

The Click-iT EdU Microplate Assay is optimized for microplate-based fluorescence analysis with a simple and rapid workflow.

In this assay the modified thymidine analog 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 fast and accurate, with reduced wash steps and a 25% (or more) time-savings advantage over traditional BrdU colorimetric or fluorescent cell proliferation assays.

The versatile red-fluorescent Amplex UltraRed reagent is utilized for the readout, which can be detected by either fluorescence or absorbance.

Graph of decreasing DNA synthesis as detected by Click-iT EdU microplate assay

Detection of proliferation using the Click-iT EdU microplate assay with aphidicolin-treated cells.

Metabolic indicators

Our metabolic indicators, which include resazurin, measure cellular reduction potential and are compatible with fluorescence- or absorption-based microplate readers. The signal emitted is proportional to the number of live cells in the well.

A choice of reporters is available and in each case, actively respiring cells reduce the indicator reagent to a strongly fluorescent or pigmented product. Nonviable or damaged cells—which are also likely to be non-proliferating—have decreased reductive capacity and thus generate a lower signal. The signal can be used to assess the average proliferation rate of a cell population when the assay is performed at multiple time points.

The alamarBlue and PrestoBlue Cell Viability Reagents are non-toxic and can be used on live cells, enabling downstream functional analyses. The alamarBlue and PrestoBlue assays are designed to be simple and convenient by providing the reagents in a proprietary stabilizing formulation with a "add, incubate, and read" protocol that is scalable from single wells to high-throughput screening (HTS). The alamarBlue HS (Cat. No. A50100 and Cat. No. A50101) and PrestoBlue HS Cell Viability Reagents (Cat. No. P50200 and Cat. No. P50201) contain highly purified resazurin and provide superior performance when compared to our first-generation alamarBlue and PrestoBlue Cell Viability Reagents.

Line graph showing growth inhibition as indicated by alamarBlue reagent

Monitoring the efficacy of a growth inhibitor with alamarBlue indicator.

DNA Content

CyQUANT cell proliferation assays provide an accurate microplate–based fluorescence method for counting cells in a population, based on cellular DNA content. Because cellular DNA content is highly regulated, the CyQUANT assay can be used at multiple time points to calculate the average proliferation rate of a cell population. Binding of the CyQUANT dye to DNA is independent of metabolic state, so signal windows and fluorescence intensities can be compared across a range of conditions and cell types.

A variety of CyQUANT assay formats provide options for different workflows, including multi-day and endpoint assays for total cell numbers and assays for live cells.

CyQUANT Cell Proliferation Assay Kit proliferation graph

Quantitation of NIH 3T3 fibroblasts using the CyQUANT Cell Proliferation Assay Kit.

Selection Guides for Imaging Cell Proliferation

The Invitrogen Click-iT Plus EdU Assay Kits provide simple, robust methods for analyzing DNA replication in proliferating cells in as little as 90 minutes. Because of the new picolyl azide and copper protectant reagents, the Click-iT Plus kits allow multiplexing with GFP, RFP, mCherry, R-PE, and R-PE tandems. Kits are available for microscopic imaging or HCS with a choice of fluorescent wavelengths for multiplexing.

  Click-iT Plus EdU Alexa Fluor 488 Imaging Kit Click-iT Plus EdU Alexa Fluor 555 Imaging Kit Click-iT Plus EdU Alexa Fluor 594 Imaging Kit Click-iT Plus EdU Alexa Fluor 647 Imaging Kit
Basis of assay The modified thymidine analog EdU is incorporated into newly synthesized DNA in proliferating cells. After a brief “click” reaction a biotin moiety is attached to the incorporated EdU, streptavidin-peroxidase attaches to the biotin and upon the addition of a chromagen substrate a dark brown signal results.
Readout Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period.
Fluorescent label Alexa Fluor 488 picolyl azide Alexa Fluor 555 picolyl azide Alexa Fluor 594 picolyl azide Alexa Fluor 647 picolyl azide
Standard filter set FITC TRITC Texas Red Cy®5
Ex/Em (nm) 495/519 555/565 590/617 650
Signal-to-noise ratio five stars five stars five stars five stars
Photostability four stars five stars five stars five stars
Bibliography
Protocol
Multiplexing The mild click chemistry protocol does not destroy cellular epitopes (unlike BrdU protocols) and is compatible with antibody labeling, fluorescent proteins, and common cell or tissue staining methods for imaging or HCS.
Sample type Optimized for live cells and tissue sections; the click detection step comes after fixation.
Format 1 kit/50–250 coverslips 1 kit/50–250 coverslips 1 kit/50–250 coverslips 1 kit/50–250 coverslips
Cat. No. C10637 C10638 C10639 C10640
Cy is a registered trademark of GE Healthcare.

Invitrogen Click-iT EdU Assay Kits provide simple, robust methods for analyzing DNA replication in proliferating cells in as little as 90 minutes. Because of the high labeling efficiency and the lack of harsh treatments, the Click-iT EdU Assays provide a superior alternative to traditional proliferation assays. The use of Invitrogen Alexa Fluor dyes results in a bright, photostable fluorescent signal designed for easy detection of proliferating cells.

  Click-iT EdU Alexa Fluor 488 Imaging Kit Click-iT EdU Alexa Fluor 555 Imaging Kit Click-iT EdU Alexa Fluor 594 Imaging Kit Click-iT EdU Alexa Fluor 647 Imaging Kit
Basis of assay The modified thymidine analog EdU is incorporated into newly synthesized DNA in proliferating cells. It labels the cells with a bright, photostable Alexa Fluor dye in a fast, highly specific click reaction.
Readout Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period.
Fluorescent label Alexa Fluor 488 Alexa Fluor 555 Alexa Fluor 594 Alexa Fluor 647
Standard filter set FITC TRITC Texas Red Cy®5
Ex/Em (nm) 495/519 555/565 590/617 650/668
Signal-to-noise ratio five stars five stars five stars five stars
Photostability four stars five stars five stars five stars
Bibliography
Protocol
Multiplexing Limited multiplexibility with fluorescent proteins.
Sample type Optimized for live cells; the click detection step comes after fixation.
Format 1 kit/50–250 coverslips 1 kit/50–250 coverslips 1 kit/50–250 coverslips 1 kit/50–250 coverslips
Cat. No. C10337 C10338 C10339 C10340
Cy is a registered trademark of GE Healthcare.

The Invitrogen Click-iT EdU Colorimetric IHC Detection Kit provides a simple, robust method for analyzing DNA replication in proliferating cells in as little as 90 minutes. Optimized for IHC (immunohistochemical) detection of proliferating cells in tissue sections. Colorimetric signal is perfect for tissues with high background fluorescence; the proliferation signal that can be archived for future analysis.

  Click-iT EdU Colorimetric IHC Detection Kit
Basis of assay The modified thymidine analog EdU is incorporated into newly synthesized DNA in proliferating cells.  After a brief “click” reaction a biotin moiety is attached to the incorporated EdU, streptavidin-peroxidase attaches to the biotin and upon the addition of a chromagen substrate a dark brown signal results.
Readout The peroxidase enzyme and subsequent colorimetric precipitation accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period.
Label Colorimetric detection
Platform Light microscopy
Bibliography
Protocol
Multiplexing Commonly used secondary tissue stains such as hematoxylin or methyl green can be used to review the cellular context surrounding the proliferation signal.
Sample type Optimized for tissue sections
Format 1 kit/50 slides
Cat. No. C10644
  Click-iT EdU Alexa Fluor 488 HCS Assay Click-iT EdU Alexa Fluor 555 HCS Assay Click-iT EdU Alexa Fluor 594 HCS Assay Click-iT EdU Alexa Fluor 647 HCS Assay
Basis of Assay The modified thymidine analog EdU is incorporated into newly synthesized DNA in proliferating cells. It labels the cells with a bright, photostable Alexa Fluor dye in a fast, highly specific click reaction.
Readout Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period.
Fluorescent Label Alexa Fluor 488 Alexa Fluor 555 Alexa Fluor 594 Alexa Fluor 647
Standard Filter Set FITC TRITC Texas Red Cy®5
Ex/Em (nm) 495/519 555/565 590/617 650/668
Signal-to-noise ratio five stars five stars five stars five stars
Photostability four stars five stars five stars five stars
Bibliography
Protocol
Multiplexing The mild click chemistry protocol does not destroy cellular epitopes (unlike BrdU protocols) and is compatible with antibody labeling and common cell- or tissue-staining methods.
Sample type Optimized for plate-based HCS assays. Reagents can be added directly to culture medium.
Format 10 plates 10 plates 10 plates 10 plates
Cat. No. C10351 C10353 C10355 C10357
Cy is a registered trademark of GE Healthcare.
Multiplex-enhanced

The Invitrogen Click-iT Plus EdU Assay Kits provide simple, robust methods for analyzing DNA replication in proliferating cells in as little as 90 minutes. Because of the new picolyl azide and copper protectant reagents, the Click-iT Plus kits allow multiplexing with GFP, RFP, mCherry, R-PE, and R-PE tandems. Kits are available for microscopic imaging or HCS with a choice of fluorescent wavelengths for multiplexing.

  Click-iT Plus EdU Alexa Fluor 488 Imaging Kit Click-iT Plus EdU Alexa Fluor 555 Imaging Kit Click-iT Plus EdU Alexa Fluor 594 Imaging Kit Click-iT Plus EdU Alexa Fluor 647 Imaging Kit
Basis of assay The modified thymidine analog EdU is incorporated into newly synthesized DNA in proliferating cells. After a brief “click” reaction a biotin moiety is attached to the incorporated EdU, streptavidin-peroxidase attaches to the biotin and upon the addition of a chromagen substrate a dark brown signal results.
Readout Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period.
Fluorescent label Alexa Fluor 488 picolyl azide Alexa Fluor 555 picolyl azide Alexa Fluor 594 picolyl azide Alexa Fluor 647 picolyl azide
Standard filter set FITC TRITC Texas Red Cy®5
Ex/Em (nm) 495/519 555/565 590/617 650
Signal-to-noise ratio five stars five stars five stars five stars
Photostability four stars five stars five stars five stars
Bibliography
Protocol
Multiplexing The mild click chemistry protocol does not destroy cellular epitopes (unlike BrdU protocols) and is compatible with antibody labeling, fluorescent proteins, and common cell or tissue staining methods for imaging or HCS.
Sample type Optimized for live cells and tissue sections; the click detection step comes after fixation.
Format 1 kit/50–250 coverslips 1 kit/50–250 coverslips 1 kit/50–250 coverslips 1 kit/50–250 coverslips
Cat. No. C10637 C10638 C10639 C10640
Cy is a registered trademark of GE Healthcare.
Fluorescence

Invitrogen Click-iT EdU Assay Kits provide simple, robust methods for analyzing DNA replication in proliferating cells in as little as 90 minutes. Because of the high labeling efficiency and the lack of harsh treatments, the Click-iT EdU Assays provide a superior alternative to traditional proliferation assays. The use of Invitrogen Alexa Fluor dyes results in a bright, photostable fluorescent signal designed for easy detection of proliferating cells.

  Click-iT EdU Alexa Fluor 488 Imaging Kit Click-iT EdU Alexa Fluor 555 Imaging Kit Click-iT EdU Alexa Fluor 594 Imaging Kit Click-iT EdU Alexa Fluor 647 Imaging Kit
Basis of assay The modified thymidine analog EdU is incorporated into newly synthesized DNA in proliferating cells. It labels the cells with a bright, photostable Alexa Fluor dye in a fast, highly specific click reaction.
Readout Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period.
Fluorescent label Alexa Fluor 488 Alexa Fluor 555 Alexa Fluor 594 Alexa Fluor 647
Standard filter set FITC TRITC Texas Red Cy®5
Ex/Em (nm) 495/519 555/565 590/617 650/668
Signal-to-noise ratio five stars five stars five stars five stars
Photostability four stars five stars five stars five stars
Bibliography
Protocol
Multiplexing Limited multiplexibility with fluorescent proteins.
Sample type Optimized for live cells; the click detection step comes after fixation.
Format 1 kit/50–250 coverslips 1 kit/50–250 coverslips 1 kit/50–250 coverslips 1 kit/50–250 coverslips
Cat. No. C10337 C10338 C10339 C10340
Cy is a registered trademark of GE Healthcare.
Colorimetric

The Invitrogen Click-iT EdU Colorimetric IHC Detection Kit provides a simple, robust method for analyzing DNA replication in proliferating cells in as little as 90 minutes. Optimized for IHC (immunohistochemical) detection of proliferating cells in tissue sections. Colorimetric signal is perfect for tissues with high background fluorescence; the proliferation signal that can be archived for future analysis.

  Click-iT EdU Colorimetric IHC Detection Kit
Basis of assay The modified thymidine analog EdU is incorporated into newly synthesized DNA in proliferating cells.  After a brief “click” reaction a biotin moiety is attached to the incorporated EdU, streptavidin-peroxidase attaches to the biotin and upon the addition of a chromagen substrate a dark brown signal results.
Readout The peroxidase enzyme and subsequent colorimetric precipitation accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period.
Label Colorimetric detection
Platform Light microscopy
Bibliography
Protocol
Multiplexing Commonly used secondary tissue stains such as hematoxylin or methyl green can be used to review the cellular context surrounding the proliferation signal.
Sample type Optimized for tissue sections
Format 1 kit/50 slides
Cat. No. C10644
Microplate-based
  Click-iT EdU Alexa Fluor 488 HCS Assay Click-iT EdU Alexa Fluor 555 HCS Assay Click-iT EdU Alexa Fluor 594 HCS Assay Click-iT EdU Alexa Fluor 647 HCS Assay
Basis of Assay The modified thymidine analog EdU is incorporated into newly synthesized DNA in proliferating cells. It labels the cells with a bright, photostable Alexa Fluor dye in a fast, highly specific click reaction.
Readout Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period.
Fluorescent Label Alexa Fluor 488 Alexa Fluor 555 Alexa Fluor 594 Alexa Fluor 647
Standard Filter Set FITC TRITC Texas Red Cy®5
Ex/Em (nm) 495/519 555/565 590/617 650/668
Signal-to-noise ratio five stars five stars five stars five stars
Photostability four stars five stars five stars five stars
Bibliography
Protocol
Multiplexing The mild click chemistry protocol does not destroy cellular epitopes (unlike BrdU protocols) and is compatible with antibody labeling and common cell- or tissue-staining methods.
Sample type Optimized for plate-based HCS assays. Reagents can be added directly to culture medium.
Format 10 plates 10 plates 10 plates 10 plates
Cat. No. C10351 C10353 C10355 C10357
Cy is a registered trademark of GE Healthcare.

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