Bridging cells of three colors with two bio-orthogonal click reactions.

Cell-cell interactions play a crucial role in the development and function of multicellular organisms. To study cell-cell interactions , it is a big challenge for researchers to artificially build up cell junctions to bridge different types of cells for this purpose. Herein, by employing two orthogonal click reactions, we rationally designed four click reagents , , , and and successfully applied them to bridge cells of three colors. Orthogonality between these two click reactions was validated in solution and characterized with HPLC and ESI-MS analyses. After modifications of fluorescent protein-expressing prokaryotic () cells (or eukaryotic HEK 293T cells) of three colors with the reagents , and , or , the cells were sequentially bridged. The HEK 293T cells showed a higher efficiency of cell bridging than the cells. Finally, using optical tweezers, we quantitatively measured the bridging probability between -modified and -modified HEK 293 cells, as well as the rupture force between two bridged cells. We found that the CBT-Cys click reaction markedly improved the efficiency of cell bridging and the rupture force between two bridged cells was measured to be 153.8 pN at a force-loading rate of 49 pN s. Our results demonstrate that it is possible to use two (or ) orthogonal click reactions to bridge three (or + 1) types of cells. Taking the biological importance of cell junctions into consideration, we anticipate that our method of bridging three types of cells with two bio-orthogonal click reactions will be a useful tool for biologists to study cell-cell interactions with more convenience and efficiency.