ClickTech EdU T Cell Proliferation Kit 647 for Flow Cytometry
EdU T Cell Proliferation Kit for Flow Cytometry
- Catalog No.
- 1 Kit / 48 assays
- € 125,00
- 1 Kit / 192 assays
- € 350,00
The detection of T cell proliferation is of utmost importance in immunology research, as it is a major indicator of T cell activation. This is normally performed by dilution-based methods, like the use of fluorescent cell tracing reagents. This approach 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. A superior alternative is the incorporation of detectable nucleoside analogues like 5-ethynyl-2’deoxyuridine (EdU) over the course of 2 hours before detection. The major advantages of our ClickTech EdU T cell proliferation assay are that this method is less cytotoxic, offers superior signal-to-background ratio and allows for improved detection of interferon gamma responses.
F.C. Stempels et al., Journal of Immunological Methods, Volume 502, 2022, 113228,
What type of cells can incorporate EdU?
The EdU cell proliferation assay has been applied to many different cell types and organism. Cell lines like HeLa, HEK, MCF-7, MOLM are arguably among the most commonly used type of samples, but it can be applied also to animals, like mouse, rat, the nematode C. elegans, crickets (Gryllus bimaculatus), chicken (Gallus domesticus) and zebra fish (Danio rerio) or even plants (e.g. Arabidopsis thaliana). Cells that possess a thymidine salvage pathway can phosphorylate EdU to the corresponding triphosphate, which is then incorporated into DNA by host DNA polymerases during replication and therefore they are compatible with the EdU assay.
How does EdU labeling compare to methods based on label dilution?
Mostly, dilution-based T cell proliferation probes conjugate unspecifically to amine groups in cells. Each cell division then results in dilution (takes several days) of the probes. Main disadvantages are cytotoxicity, an impact on T cell activation and long incubation time (several days and up to a week) before readout. The superior EdU cell proliferation assay relies on the incorporation of the modified uridine analog EdU in the DNA of proliferating T cells (just 2 hours before harvesting) and subsequent detection. Both methods enable detection and quantification of T cell proliferation by flow cytometry. Our EdU incorporation assay is very sensitive, and can be multiplexed with other fluorescent stainings.
- EdU outperforms a standard dilution-based probe in detecting T cell proliferation
- The EdU assay is less cytotoxic for human T cells
- The EdU assay offers superior signal-to-background ratio
- The EdU assay allows for better discernable interferon gamma responses
Can I combine the EdU T cell Proliferation assay and staining with PerCP, APC, APC-based tandems, RPE, PR-tandem, Quantum Dot antibody conjugates or intracellular antigens?
Yes, this is feasible. Please note that staining with PerCP, APC and APC-based tandems may be performed before the EdU detection step (click reaction), while RPE, PR-tandem or Quantum Dot antibody conjugates and detection of intracellular antigens should be performed after the click reaction. Check also the user manual for further information. Please, make sure that the emission spectra of the fluorochromes to be combined do not overlap extensively, so that their signals can be distinguished unequivocally using appropriate emission filters or spectral demixing.
When may break points be introduced during the protocol?
It is possible to safely take a break after the fixation step. Remove the fixation solution, wash as suggested in the user manual and store the cells in wash buffer at 4° C. The permeabilization step is not required immediately. However, the protocol may also be safely paused after permeabilization, following the same recommendation as above.
It is important to not pause the protocol if the click cocktail for EdU detection has been prepared, since the cocktail reacts optimally within 15 minutes.
What is the optimal duration of EdU pulse?
The duration of the EdU incubation step may need to be adjusted according to your cell type or organism, the proliferative state of the cells (e.g., quiescence, lymphocyte activation), the EdU concentration applied and even the experimental design. For a start it is advisable to refer to a published protocol (which is close to your experimental setup) and to test the conditions with a low number of samples. As a general guideline we recommend to use a maximal EdU concentration in cell culture medium of 10 µM for incubations of a few hours to maximum one day. For longer incubation durations the concentration should be decreased to 1-5 µM. Cells that divide rapidly (once a day) generally require shorter incubation compared to cells with lower proliferation rates. Unless specific T lymphocyte preparations are expected to be in a rather quiescent state, a 2 h pulse with 10 µM EdU is usually appropriate for activated human T lymphocytes (and likely mouse ones as well).
- What type of cells can incorporate EdU?
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