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 Molecular Probes® 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

See also flow cytometry assays 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. The Click-iT® EdU assay 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 Imaging Assays use 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.

 

Click-iT® Plus

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.

 

Click-iT® EdU microplate assay

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

Metabolic Indicators

Molecular Probes® metabolic indicators 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.

alamarBlue® indicator is nontoxic, and assays can be performed on live cells, enabling downstream functional analyses.

 

alamarBlue® Reduction

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

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

Selection Guides for Cell Proliferation Products

The 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.

Optimized for Fluorescence Microscopy

  Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit Click-iT® Plus EdU Plus 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. 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 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) 495/519 555/565 590/617 650/668
Signal-to-noise ratio five stars five stars five stars five stars
Photostability four stars five stars five stars five stars
Bibliography
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

 

Optimized for Microplate-Based HCS

  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
Multiplexing The mild click chemistry–based protocol does not destroy cellular epitopes (unlike BrdU protocols) and is compatible with antibody labeling and common cell- or tissue-staining methods for HCS. Reaqgents can be added directly to the culture medium
Format 2 plates 10 plates 2 plates 10 plates 2 plates 10 plates 2 plates 10 plates
Cat. No. C10350 C10351 C10352 C10353 C10354 C10355 C10356 C10357

Microplate assays provide proliferation information on a cell population (i.e., enumeration or total cell number) with no data on individual cells or subpopulations

 
Basis of assayDetects newly synthesized DNA (i.e., actively dividing cells)
ReadoutSignal increases with new DNA synthesis; can be detected by either fluorescence or absorbance
Fluorescent labelAmplex® UltraRed
Standard filter setRhodamine
BibliographyCitations
MultiplexingNo
Live cellsNo
Fixed cellsNo
Format1 kit/4 plates
Cat. No.C10214

Microplate assays provide proliferation information on a cell population (i.e., enumeration or total cell number) with no data on individual cells or subpopulations

  alamarBlue® Indicator XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) Vybrant® MTT Cell Proliferation Assay Kit (1,000 assays)
Basis of assayMeasures cellular reduction potential; signal is proportional to the number of live cells
ReadoutFluorescent or colorimetric signal from whole cells or lyastesColorimetric readout from cell lysates
Fluorescent labelResazurinXTT/FormazanMTT/Formazan
Fluorescence/AbsorbanceFL 571/585 nmABSABS
BibliographyCitationsCitationsCitations
MultiplexingYesNoNo
Format25 mL100 mL100 mg1 kit/10 plates
Cat. No.DAL1025DAL1100X6493V13154

Microplate assays provide proliferation information on a cell population (i.e., enumeration or total cell number) with no data on individual cells or subpopulations.

 
Basis of assayDetects cell number based on DNA content; readout is independent of metabolic state
ReadoutCultured cells must be frozen during multi-point tests and assayed together with common standardsStreamlined protocol for fast readout; eliminates freezing step; 1-hour assay from start to finish15-minute protocol; no freezing, lysis, or temperature equilibration; viability indicator (i.e., does not count dead cells)
Range50–50,000 cells100–20,000 cells50–20,000 cells
Fluorescent labelCyQUANT® GRCyQUANT® NFCyQUANT® Direct
Standard filter setFITC
BibliographyCitations
Top/bottom readBothBothBottom read
MultiplexingNoYesNo
Format1 kit/1,000 assays1 kit/200 assays1 kit/10 plates
Cat. No.C7026C35006C35011