EdU Click FC-488

The number at the end of the product name specifies the excitation wavelength of the included fluorescent marker. Suitable excitation source are:

Our ROTI^®Kits for EdU mediated detection of cell proliferation provide a superior alternative to time-consuming and less sensitive bromodeoxyuridine (BrdU) assays. Similar to BrdU assays, our assays utilise a uracil derivative (in this case ethynyldeoxyuridine, EdU) which is incorporated into the replicating DNA. EdU is not detected via antibodies; instead fluorescent dyes are coupled directly via click reaction. This fast and elegant approach allows for direct labelling of replicating DNA with the desired fluorescent dye. Thus, the assay is perfectly adaptable to the present facilities and to the assay situation (e.g. for double or triple detection). Furthermore, the click reaction is highly selective and therefore prevents any unspecific labelling. The protocol includes only few steps and decreases the total amount of time.

Kits available for:

• Fluorescence microscopy (ROTI^®Kits for Imaging)
• Flow Cytometry (ROTI^®Kits for Flow Cytometry)
• High Throughput Screening (ROTI^®Kits for High Throughput Screening)

Advantages of the click chemistry based approach:

Schematic illustration of the click chemistry based EdU cell proliferation assay. A) Incubation of cells with EdU. EdU is a thymidine analogue and is incorporated in DNA during active DNA synthesis. B) After DNA synthesis, EdU was incorporated in the DNA strands. C) Detection of proliferating cells via click chemistry. This reaction is finished within 30 minutes, whereby a variety of different fluorescent dye azides can be used.

Advantages of the Click Assay:

• Highly reliable
• Easy handling
• Fast detection procedure (only 30 min.)
• Mild conditions, no DNA denaturation required
• Compatible with various dyes
• Modular system in click chemistry
• Compatible with multiplexing

The copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) is the most prominent example of a group of reactions named click reactions. According to Sharpless’ definition, these reactions are characterised by high yields, mild reaction conditions, and by their tolerance of a broad range of functional groups.^[1-3] Typically, the reactions require simple or no workup or purification of the product. The most important characteristic of the CuAAC reaction is its unique bioorthogonality as neither azide nor terminal alkyne functional groups are generally present in natural systems.

References:
1 H. C. Kolb, M.G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed. 2001, 40, 2004-2021.
2 C.W. Tornoe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057-3064.
3 V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. 2002, 114, 2708-2711; Angew. Chem. Int. Ed. 2002, 41, 2596-2599.

The ROTI^®Kits for Flow Cytometry by Carl ROTH use click chemistry to detect cell proliferation by means of a large number of fluorescent stain read-outs. In the detection procedure both, the total number of steps and the processing time are significantly reduced. The simple click chemistry detection procedure is complete within 30 minutes.

References:
1 N. Dinh Van et al., Modulation of HCV reinfection after orthotopic liver transplantation by fibroblast growth factor-2 and other non-interferon mediators. Gut 2016, 65, 1015-1023.
2 L. Pisarsky et al., Targeting Metabolic Symbiosis to Overcome Resistance to Anti-angiogenic Therapy. Cell Reports 2016, 15, 1-14.