Cell Analysis – Cell Proliferation High-throughput Screening 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 HTS

The baseclick ClickTech EdU cell Proliferation assays overcome the limitations of conventional cell proliferation assays and offer a superior alternative to BrdU and [3H] thymidine assays. EdU (5-ethynyl-2′-deoxyuridine) is an alkyne-modified thymidine analog, which is incorporated into DNA during active DNA synthesis.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). The optimized detection protocol considerably simplifies the workflow and the time required. The simple detection procedure is completed within 30 minutes and is compatible with multiplexing for content- and context-rich results.

Workflow

Application

High Throughput Screening (HTS) is widely used in the pharmaceutical industry for drug discovery purposes. It is used to simultaneously study the effects of large numbers of compounds such as ligands (for receptors, ion channels or other pharmacological targets) on cell proliferation/DNA synthesis or toxicity. The baseclick EdU HTS kits measure DNA synthesis of adherent cells in 96 well plates and enable multiplexing with a variety of fluorescent dyes.

EdU incorporation over time and increasing cell numbers. HeLa cells were seeded at the indicated densities in a 96 well plate and were incubated with 10 µM EdU for 2, 4 and 6 hrs. EdU incorporation was detected using the BCK-EdU555HTS Kit.

Product Overview

LITERATURE

Establishment of long-term ostracod epidermal culture, S. Morgan et al., 2020, In Vitro Cell. Dev. Biol. Anim., Vol. 56, p. 760-772.

https://doi.org/10.1007/s11626-020-00508-8

Exposure of vital cells to necrotic cell lysates induce the IRE1α branch of the unfolded protein response and cell proliferation, P. Rohne et al., 2017, Cell Stress and Chaperones, Vol. 23, p. 77-89.

https://doi.org/10.1007/s12192-017-0825-6

Satellite DNA in Vicia faba is characterized by remarkable diversity in its sequence composition, association with centromeres, and replication timing, L. Robledillo et al., 2018, Scientific Reports, Vol. 8, p. 5838.

https://doi.org/10.1038/s41598-018-24196-3

LINC00319 acts as a microRNA-335–5p sponge to accelerate tumor growth and metastasis in gastric cancer by upregulating ADCY3, J. Zou et al., 2019, American Journal of Physiology-Gastrointestinal and Liver Physiology, Vol. 318(1), G10-G22.

https://doi.org/10.1152/ajpgi.00405.2018