3-D reconstruction of HA-Srt dynamics on the cell surface.

<p>(<b>A</b>) <b>Real time imaging of the disappearance of HA-strep-Qdot655 from the cell surface.</b> MDCK cells were infected with HA-Srt, labeled with 100 µM sortase and 250 µM biotin probe followed by 20 nmQdot655 as described. Images were acquired every three minutes in Z-stack series of 8 µm in 21 images. Imaris software was used to create 3D images. Top figure displays the entire frame while the bottom image is focused on a single cell. Scale bars = 5 µm. (<b>B</b>) <b>Real time imaging of CD154/CD40L on the cell surface.</b> CD154/CD40L expressing MDCK cells were labeled with 100 µM sortase and 250 µM biotin probe and subsequently with 10 nm Qdot 655. Images were acquired every three minutes in Z-stack series of 8 µm in 21 images. Imaris software was used to create 3D images. Top figure displays the entire frame while the bottom image is focused on a single cell. Scale bars = 5 µm. (<b>C</b>) <b>Sum of pixel intensities in 7A and 7B.</b> The sum of pixel intensities at every timepoint was analyzed with imaris software and percent reductions quantified relative to the first timepoint.