Cell analysis – T Cell Proliferation Assays for Flow Cytometry

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 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.

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.

ClickTech EdU T Cell Proliferation Assays for FC

ClickTech EdU T cell Proliferation assay enables monitoring genotoxicity, anticancer drug evaluation or cell cycle analysis by direct detection of DNA replication in proliferating cells. 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. This nucleoside contains a reactive alkyne group (instead of the bromine atom in BrdU) and can be conjugated to molecules bearing azide groups through 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 conditions and are easy to perform. In order to start the 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.

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. baseclick currently offers EdU T Cell proliferation assays for flow cytometry and three standard dyes (488, 555, 647 nm).

Workflow

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

Application

Comparison between the BrdU and the EdU assay:

Comparison between cell tracing and the EdU assay:

Fluorescent cell tracing reagents exhibits several limitations as it can reduce T cell proliferation, viability, responsiveness, and cell proliferation can only be determined after completion of full rounds of cell division.

Stimulation of human T cell activation via comparing the dilution-based T cell proliferation probe CellTrace Far Red and EdU. ClickTech EdU T cell proliferation Kit overcomes the limitations of dilution-based probes, thus providing a superior alternative for measuring T cell proliferation.

Although both the ClickTech EdU Cell Proliferation assays and fluorescent cell tracing assays such as CellTraceTM are used to estimate cell proliferation, the actual information they provide is different as they measure distinct parameters/aspects of cell proliferation:

• By measuring dilution of a signal through cell division, fluorescent cell tracing assays give an estimate of how many cell generations have taken place between the labelling pulse with the tracing reagent and the time at which signal detection by FACS is performed (usually 4-5 days).
• In contrast, by determining the fraction of cells replicating their DNA, the ClickTech EdU T cell Proliferation assay estimates the proliferation rate of a T cell population at the time of the EdU pulse, which in the standard application of the assay is immediately before detection. Changes in the proliferation rate of a T cell population over a prolonged period can also be monitored by performing the ClickTech EdU T cell Proliferation assay at different time points during that period. Information on the number of cell generations that take place over a period can also be obtained by applying a single, initial EdU pulse on a cell population and performing EdU detection on aliquots of the same cell population not only immediately after the EdU pulse, but also at later time points after washout of the pulse (a so-called chase). Here too, the decrease of signal intensity allows estimating how many cell divisions have occurred since the initial pulse. Thus, the ClickTech EdU T cell Proliferation assay is more versatile than fluorescent cell tracing assays.

LITERATURE

A sensitive and less cytotoxic assay for identification of proliferating T cells based on bioorthogonally-functionalized uridine analogue, F. C. Stempels et al., 2022, Journal of Immunological Methods, Vol. 502,  p. 113228.
https://doi.org/10.1016/j.jim.2022.113228