• Highly reliable
• Easy to perform
• Fast detection procedure (only 30 min)
• Mild conditions (no DNA denaturation required)
• Modular system due to click chemistry
• Compatible with various dyes and readouts
• Compatible with multiplexing
• No cytotoxicity up to 1 mM EdU

The ROTI^®kits for Flow Cytometry allow the measurement of DNA synthesis in flow cytometry. 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. The protocol includes only few steps and decreases the total amount of time.

Figure: HeLa cells, treated without (A) and with (B) 10µM EdU for 2 h. Click reaction was performed using 6-FAM-Azide. A: negative control, cells without EdU incorporation. B: non-proliferating cells without EdU incorporation (blue coloured left peak) and proliferating cells (S-phase) having incorporated EdU and are labeled with 6-FAM Azide (green coloured right peak). Density blots of PI stained samples without (C) and with (D) EdU incubation and followed click reaction. The y-axis presents the FL1-Fluorescence intensity and the x-axis the content of DNA measured with FL3-area. Cell cycle phases are indicated as G1, S and G2/M phase.

The number at the end of the product name specifies the wavelength at which the included fluorescent marker is optimally excited.