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. Author manuscript; available in PMC: 2022 May 6.
Published in final edited form as: J Vis Exp. 2021 Jun 1;(172):10.3791/62559. doi: 10.3791/62559

Figure 3: Bead-loaded cells remain adherent and are healthy enough to grow and divide.

Figure 3:

(A) U2OS cells were bead-loaded with 0.5 μg of Cy3-conjugated anti-FLAG Fab in 4 μL of bead loading solution. The cells were imaged directly before, directly after bead loading, and 24 h after bead loading. Orange arrows identify areas where cells peeled off during bead loading. Scale bars = 2 mm. (B) Representative image of U2OS cells bead-loaded with components from (A) but with harsh tapping and too many beads. The red arrow identifies extra glass beads. Scale bar = 2 mm. (C) U2OS cells were loaded with 1.5 μg of the 14.4 kbp plasmid smFLAG-KDM5B-15xBoxB-24xMS2, 0.5 μg of Cy3-conjugated anti-FLAG Fab (green), 130 ng of HaloTag-MCP (magenta) in 8 μL of bead loading solution. Directly before imaging, the HaloTag was stained with JF646-HaloLigand. The MS2 stem-loops of the mRNA transcribed from the reporter plasmid are labeled by MCP (magenta spots), and FLAG-tagged translated reporter protein is labeled by anti-FLAG Fab (green colocalization to mRNA). Mature Fab-labeled protein localizes to the nucleus. This cell was imaged 4-15 h after bead loading. Yellow arrows identify the cell nucleus before and nuclei after cell division. Scale bars = 5 μm. Abbreviation: MCP = MS2 coat protein. Please click here to view a larger version of this figure.