Living cell laser scanning confocal microscope

Hela cells (ATCC CCL-2) were seeded into 8-well Nunc Lab-Tek confocal imaging plate with 70−80% confluency. The cells were then transfected with 125 ng pcDNA3.1-s-ACE2 vector per well using Lipofectamine 3000. 24 h later, the cells were incubated with the fresh medium containing 500 nM SNAP-Cy3, 25 nM h-RBD-SiR, and 1 μM Hoechst 33342. After incubation for 1 h at 37°C in the presence of 5% CO_2, the fluorescence imaging was performed with 100 × oil-dripping objective lens by using ANDOR™ living cell laser scanning confocal microscope. For SNAP-Cy3, λ_ex = 561 nm, λ_em = 580-650 nm; For Halo-SiR, λ_ex = 640 nm, λ_em = 660-700 nm.

Hela and HEK293T cells seeded in 8-well confocal imaging plates were transfected with 125 ng pcDNA3.1-s-ACE2 vector. After transfection for 24 h, the first group cells were treated with 250 nM SNAP-Alexa 488 only, the second group cells were treated with 250 nM SNAP-Alexa 488 and 25 nM h-RBD-SiR, and the third group cells were pretreated with 50 μM dynasore for 30 min, then incubated with 250 nM SNAP-Alexa 488 and 25 nM h-RBD-SiR. The fluorescence imaging was acquired every 10 min between 20 and 100 min. The imaging was performed with 100 × oil-dripping objective lens by using ANDOR™ living cell laser scanning confocal microscope. For s-ACE2-488, λ_ex = 488 nm, λ_em = 500-550 nm; For h-RBD-SiR, λ_ex = 640 nm, λ_em = 660-700 nm.