In the Click-i T® Plus Ed U assay, the alkyne-containing thymidine analog Ed U (5-ethynyl-2´-deoxyuridine) is incorporated into DNA during active DNA synthesis .
The incorporated Ed U is then detected by a click reaction—a copper-catalyzed azide–alkyne cycloaddition—using a fluorescent Alexa Fluor® or Pacific Blue™ dye containing a picolyl azide moiety (Figure 2B).
The denaturants also limit the ability of the Brd U assay to be multiplexed with fluorescent proteins (e.g., GFP, RFP, m Cherry) or phycobiliproteins (e.g., R-PE, R-PE tandems), which are regularly used in imaging or flow cytometry applications.
Unlike these traditional cell proliferation assays, the Click‑i T® Plus Ed U proliferation assay does not rely on radioactivity or antibodies for detection of the newly incorporated deoxyribonucleoside.
The harsh treatment required for the antibody-based Brd U assay resulted in the loss of the GFP signal, as seen by the absence of green fluorescence in Figures 4A and 4B.
Furthermore, the Brd U proliferation signal required a 10-fold longer exposure time (Figure 4B) to generate results comparable to those obtained with the Click-i T® Plus Ed U assay (Figure 4C). Cell proliferation detected using the Brd U assay or the Click-i T® Plus Ed U assay.
Figure 7 shows co-labeling of Jurkat cells with Click-i T® Plus Alexa Fluor® 488 picolyl azide to detect incorporated Ed U and with PE-Cy®7–conjugated mouse anti–human CD4 antibody.
Although it eliminates the complications of working with radioactivity, the Brd U proliferation assay is both difficult to perform consistently and time consuming, typically requiring 6–24 hr to complete.(See a list of the products featured in this article.) Cell proliferation assays provide a critical piece of the puzzle when evaluating cell health, genotoxicity, and the efficacy of anti-cancer drugs.Proliferation, however, is rarely assayed in isolation; other cell function probes are often used in concert with proliferation assays to provide a more informative picture of the state of the cell.Both cell samples were treated with Hoechst® 33342 nucleic acid stain (blue); high-content analysis was performed using the Thermo Scientific® Cellomics® Arrray Scan® VTI HCS Reader.The ability to multiplex Click-i T® Plus Ed U assays with other fluorescent probes opens the door to a more complete analysis of cell function.