- After SPAAC staining (30 min at 37C), remove the media and
add 1 mL of Hoechst 33342 (1μM) prepared in complete
DMEM to the cells. Place the dish immediately in the incuba-
tor at 37C for 30 min. - Wash the cells once with 1 mL of live cell imaging medium
prepared as mentioned in Subheading2.2. - Add 1 mL of live cell imaging medium and image the cells
using a confocal laser scanning microscope with an oil immer-
sion at 63(seeNotes 12and 14 , Fig.5).
Fig. 5Imaging cellular RNA transcription using AMUTP under copper-free SPAAC
reaction conditions in live HeLa cells. The AMU-labeled RNA transcripts in live
cells were detected by treating the cells with Cy3 DBCO. This figure has been
reproduced by permission of Nucleic Acids Research: Oxford Journals [24]368 Anupam A. Sawant et al.