There are many different types of cell proliferation assays. The assay you choose depends on the number and type of cells that you are studying and the mechanism of action you want to study. Select from our broad range of assays for both flow cytometry and imaging (including microplate and HCS) applications.
Cell Proliferation Assay Protocols Cell Cycle & Proliferation Pathways
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Cell proliferation assays selection guide
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| Assays and kits | Click-iT EdU and BrdU Assays | CellTrace and CFSE reagents | CyQUANT Cell Proliferation kits | Resazurin (alamarBlue, PrestoBlue) | CyQUANT MTT and XTT |
| Mechanism | New DNA synthesis | Generational analysis by dye dilution | Changes in DNA content quantitation | Changes in cellular metabolism, metabolic indicators | |
| How cell proliferation is measured | Detection of thymidine analog incorporated in newly synthesized DNA during cell proliferation End point assays Cells or tissue | Live cell analysis of cells permanently labeled with non-toxic stain Live cell, multi-day analysis Kinetic assay Cells | Nucleic acid binding stains, primarily DNA End point assays Fixed or live cells | Metabolic activity of cell turns over the substrate (resazurin) which can be read by absorbance or fluorescence Kinetic assay | Metabolic activity of cell turns over the substrate which can be read by absorbance End point assay |
| What is being measured | Cells proliferating in a window of time that the thymidine nucleoside analog EdU or BrdU is present (and when drug of choice is testing cells) | Number of generations (cell doublings) after time point zero, where labeling dye was introduced | Nucleic acid content in cells against a standard curve of known cell number | Measure cellular reduction potential Signal is proportional to the number of live, metabolizing cells | Measure cellular reduction potential Signal is proportional to the number of live, metabolizing cells |
| Detection platform | Flow cytometry Imaging (includes HCS and microplate reader) | Flow cytometry | Microplate reader | Microplate reader | Microplate reader |
DNA synthesis cell proliferation assays
Measuring the synthesis of new DNA is a precise way to assay cell proliferation in individual cells or in cell populations. The rate of new DNA synthesis can be based on incorporation of a nucleoside analog such as BrdU or EdU into DNA. Detection of these two thymidine analogs varies widely and has implications on the assay results and multiplexability.
See a selection guide and learn about Click-iT EdU assays and how they compare to traditional BrdU assay methods
Multicolor imaging of HeLa cells with Click-iT Plus EdU Alexa Fluor 647 Imaging Kit.
Results from immunophenotyping experiment to evaluate CD3 and DNA strand breaks in human T cells. Dual parameter plot of Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit and Hu CD3 PE-Cy7 fluorescence.
Generational analysis by dye dilution
By permanently labeling cells with a fluorescent stain, without affecting morphology or physiology, it is possible to trace generations of cell division by flow cytometry. The cell tracing dye CFSE has historically been used in cell proliferation studies, but it is not without its limitations. With the development of CFSE alternatives such as CellTrace Violet that do not require 488 nm excitation, cell proliferation assays are now more amenable to multiplex analyses with other fluorescent probes. Additionally, many of the CFSE alternatives, the CellTrace reagents, are not only less toxic than CFSE, but also allow for more generations to be visualized when compared to CFSE due to bright, single-peak staining.
Learn more about CellTrace reagents as improved CFSE alternatives
Human peripheral blood lymphocytes were harvested and stained with CellTrace Violet and analyzed by flow cytometry.
DNA content quantitation cell proliferation assays
Quantitation of NIH 3T3 fibroblasts using the CyQUANT Cell Proliferation Assay Kit.
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, CyQUANT assays 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.
Learn more about CyQUANT Cell Proliferation Assays
Metabolic indicators as cell proliferation assays
Metabolic indicators such as resazurin measure cellular reduction potential and are compatible with fluorescence- or absorbance-based microplate readers. The signal emitted is proportional to the number of live cells in the well. alamarBlue is recommended in cases of extended viability studies or when using a high cell density in microplate assays, and PrestoBlue is recommended for quick viability determination in microplate assays (10 minute incubation).
A colorimetric option is also available for microplate readers. Actively respiring cells reduce the indicator reagents, MTT or XTT, to pigmented products. Nonviable or damaged cells—which are also likely to be non-proliferating—have decreased reductive capacity and thus generate a lower signal. Although often used to determine cell viability of a population from a single time point, the MTT and XTT signals can be used to assess the average proliferation rate of a cell population when the assay is performed at multiple time points.
Learn more about Microplate Assays for Cell Viability
Easy multiplexing of a caspase-3/7 detection reagent and PrestoBlue Cell Viability Reagent using high-throughput screening.
Immunology at work
Learn immune cell type markers and protocols. The Invitrogen Immunology at Work Resource Center is a learning center with technical content designed for new and experienced life scientists alike exploring the field of immunology.
Resources
Tools
Flow Cytometry Panel Builder—Design your flow cytometry panel with this online tool for a simplified, customizable experience to fit your needs.
Fluorescence SpectraViewer—Online tool for visualization of the excitation and emission of fluorescent reagents. Tool allows for checking spectral compatibility for multiple fluorophores.
Cell viability, proliferation, and cell cycle information—Find educational resources such as application notes, webinars, videos, articles, and more that cover the use of many of our reagents and kits for monitoring cell function.
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