A Suzuki–Miyaura method for labelling proliferating cells containing incorporated BrdU

Abstract

The 5-bromo-2′-deoxyuridine (BrdU) incorporation cell proliferation assay is the most commonly used method for assessing DNA replication. The current detection of BrdU in cells relies on antibody immunostaining, but has various limitations including low antibody specificity and poor tissue penetration. In this study, we utilised a Suzuki–Miyaura reaction to develop a chemical method to label cellular BrdU with fluorescent boronic acid probes. The coupling conditions were optimised for complex cellular environments, and the key observation was the need to use oxygen scavengers and zerovalent palladium to prevent side reactions and increase the rate of coupling. The reliability and specificity of the BrdU Suzuki–Miyaura labelling method were verified under various biological conditions. The applicability of the BrdU Suzuki–Miyaura labelling methodology was also investigated, and we show that labelling cellular BrdU is highly sensitive and reliable, which is comparable to the ideal performance of BrdU immunostaining. Moreover, the Suzuki–Miyaura reaction protocol provides high BrdU recognition specificity. Taken together, the BrdU Suzuki–Miyaura labelling protocol provides an attractive alternative to the more traditional cell proliferation assay.