Cell Analysis – Cell Proliferation Flow Cytometry Assays

Cell analysis allows scientists to gain unique insights into cell proliferation, viability, and toxicity, enabling the monitoring of cell response and cell health in cultures after treatment with various stimuli.

Detection of DNA replication is a powerful tool for studying proliferation of both cell populations and individual cells. DNA replication rates can be detected through the incorporation of a nucleoside analog such as BrdU or EdU into DNA. The detection of these two thymidine analogs differs significantly and has implications for test results, potential applications, and multiplexing capability. Compared to other cell proliferation methods, the EdU proliferation assay does not involve radioactive isotopes or antibodies to detect newly synthesized DNA.

The ClickTech EdU cell proliferation assay enables estimates of cell proliferation rates by determining the fraction of cells replicating their DNA.

The technology is based on the labeling of DNA with EdU (5-ethynyl-2′-deoxyuridine), an alkyne-modified nucleoside that is incorporated into the replicating DNA of living cells and its conjugation to a fluorochrome in situ.

Schematic representation of EdU cell proliferation assays. A) Incubation of cells with EdU. B) EdU is incorporated during active DNA synthesis. C) Detection of cell proliferation via click chemistry, wherein the use of a range of different fluorescent dyes is possible.

Advantages:

• Increased selectivity and sensibility in comparison to BrdU
• Simple, fast and reliable assay
• High sensitivity paired with very low toxicity
• A cost-effective assay
• Multiplexing possibility
• higher preservation of morphological details relative to BrdU

ClickTech EdU Cell Proliferation Assays for FC

The ClickTech EdU Cell Proliferation Kit for FC is a simple and stable assay perfectly suited for monitoring genotoxicity, anticancer drug evaluation or cell cycle analysis by directly measuring the proportion of cells that replicate their DNA. The principle of this procedure is similar to that of the BrdU assay, with the difference of the detection reaction. In summary, the thymidine analog, in this case the EdU (5-ethynyl-2′-deoxyuridine), is added to the cell culture media, taken up by the cells and incorporated into newly synthesized DNA in cell division. This nucleoside contains a reactive alkyne group (instead of the bromine atom in BrdU) and incorporated nucleotides can undergo a click reaction. After an incubation period that depends on the specific cell type, the cells are washed, fixed and permeabilized to allow the detection reagents to penetrate. The detection is then carried out via the copper-catalyzed click reaction (also known as Azide-Akyne Cyclo-addition or CuAAC). Click reactions show high quantitative yields, run under mild reaction conditions and are easy to perform. In order to start the detection reaction, a click cocktail, containing buffer, catalytic reagents and a fluorescent dye, is added to the cells . The dye contains an azide group that reacts with the alkyne group of the incorporated EdU. Only cells that actively replicate their DNA during the EdU pulse can be detected by this method.

Workflow

EdU Detection

Functional alkyne and azide groups do not occur in natural cell systems and do not react without catalytic reagents. This ensures that the EdU cell proliferation method, based on click chemistry, is highly selective, sensitive and reproducible. This technology allows an almost unlimited choice of different and easy to synthesize fluorescent dyes and thus offers maximum flexibility in terms of “detection colors” and analytical devices. Cell proliferation can be detected by fluorescence microscopy, flow cytometry and fluorescence plate reader. baseclick currently offers assays for these three analytical methods and up to four standard dyes (488, 555, 594, 647 nm).

Cell proliferation assay with the EdU Cell Proliferation Kit for Flow Cytometry (BCK-EdU488FC). HeLa cells were either untreated (A) or incubated with 10 µM EdU for 2 hours (B). The click reaction was performed using 6-FAM Azide and fluorescence intensity of 10.000 cells was measured by flow cytometry. The results are presented in form of histograms, showing the cell number in the y-axis and the FL1-Fluorescence in the x-axis.

Product Overview

LITERATURE

Cytolethal Distending Toxin Promotes Replicative Stress Leading to Genetic Instability Transmitted to Daughter Cells, W. Tremblay et al., 2021, Frontiers in Cell and Developmental Biology, Vol. 9, p. 656795.

https://doi.org/10.3389/fcell.2021.656795

Innovative DNA-Targeted Metallo-prodrug Strategy Combining Histone Deacetylase Inhibition with Oxidative Stress, T. McGivern et al., 2018, Mol. Pharmaceutics, Vol.15, p. 5058-5071.

https://doi.org/10.1021/acs.molpharmaceut.8b00652

YB-1 Expression and Phosphorylation Regulate Tumorigenicity and Invasiveness in Melanoma by Influencing EMT, C. Kosnopfel et al., 2018, Mol Cancer Res., Vol. 16, p. 1149-1160.

https://doi.org/10.1158/1541-7786.MCR-17-0528

Chronic exposure to Cytolethal Distending Toxin (CDT) promotes a cGAS-dependent type I interferon response, B. J. Pons et al., 2021, Cellular and Molecular Life Sciences, Vol. 78, p. 6319–6335.

https://doi.org/10.1007/s00018-021-03902-x