ClickTech EdU T Cell Proliferation Kit 647 for Flow Cytometry
EdU T Cell Proliferation Kit for Flow Cytometry
- Size
- Catalog No.
- Price
- 1 Kit / 48 assays
BCK-TCell-FC647_48
- € 150,00
- 1 Kit / 192 assays
BCK-TCell-FC647_192
- € 400,00
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All EdU based proliferation kits on the market are patented by our company. This click assay has been optimized for the detection of T cell proliferation. Of utmost importance in immunology research is the activation of T cells. This is normally done by BrdU assays and in special applications by fluorescent cell tracing reagents.
The used BrdU assays have several limitations as it is time consuming (4 h) and it requires harsh, denaturing conditions of tissue to allow anti-BrdU antibodies to reach the genomic DNA. Additionally the low sensitivity requires a high number of target cells.
The indirect fluorescent cell tracing reagent method depends on long incubation time (4 – 7 days) and in higher concentration shows cytotoxicity.
The EdU assay is quicker (1 h), simpler, and highly sensitive by the bio orthogonal CuAAC reaction. Therefore, EdU is a superior sensitive alternative to BrdU and the dilution methods, is less cytotoxic and allows improved detection of e.g. interferon gamma responses.
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.2022.113228FAQ
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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 interrupt the protocol after the fixation step. Thereto, remove the fixation solution and wash as suggested by the user manual, then the cells can be stored in buffer at 4° C. Alternatively, the experiment can also be safely interrupted after permeabilization, again as described above.
Please note: It is important to proceed with the experiment if the click cocktail for the detection of the EdU has been prepared already. - How to determine the EdU incubation time?
The EdU incubation time depends on the cell type or organism, the applied EdU concentration and the experimental design. For a start it is advisable to refer to a literature 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 maximum of 10 µM final EdU in the cell culture medium for incubations. For longer incubation (> 1 day) the concentration should be decreased to 1-5 µM.
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How does EdU labeling compare to methods based on label dilution?
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- Shelf Life
12 months unopened after receipt
- Storage Conditions
2-8 °C
- Physical State
kit system made of different components
- CAS Number
n.a.
- Excitation (max)
643 nm
- Emission (max)
662 nm
- Ɛ (max)
250.000 cm-1M-1
- Preparation/Handling
please see user manual of the kit
- Shelf Life