Bead Loading Proteins and Nucleic Acids into Adherent Human Cells

Abstract

Many live-cell imaging experiments use exogenous particles (e.g., peptides, antibodies, beads) to label or function within cells. However, introducing proteins into a cell across its membrane is difficult. The limited selection of current methods struggles with low efficiency, requires expensive and technically demanding equipment, or functions within narrow parameters. Here, we describe a relatively simple and costeffective technique for loading DNA, RNA, and proteins into live human cells. Bead loading induces a temporary mechanical disruption to the cell membrane, allowing macromolecules to enter adherent, live mammalian cells. At less than 0.01 USD per experiment, bead loading is the least expensive cell loading method available. Moreover, bead loading does not substantially stress cells or impact their viability or proliferation. This manuscript describes the steps of the bead loading procedure, adaptations, variations, and technical limitations. This methodology is especially suited for live-cell imaging but provides a practical solution for other applications requiring the introduction of proteins, beads, RNA, or plasmids into living, adherent mammalian cells.

Generation and diversification of recombinant monoclonal antibodies

“…We were able to purify the scFvC fragments on our affinity columns, since they contain a portion of the HC constant region that is recognized by Protein A. The purified Hec1 and Mad2-C scFvC fragments were introduced into cells by bead-loading ( McNeil and Warder, 1987 ; Cialek et al, 2021 ), and the cells were subsequently fixed and incubated with anti-rabbit secondary antibodies. As shown in Figure 5B and C , the fragments recognized kinetochores in mitotic cells, and the results were similar to those obtained with the intact, bivalent antibodies.…”

“…To inhibit Mps1 kinase, cells were treated with 10 µM reversine (Adooq Biosciences) for 1 hr prior to fixation for immunofluorescence or harvesting for immunoblot analysis. For bead loading experiments, antibodies were bead-loaded into HeLa cells 1 hr prior to fixing or imaging ( Cialek et al, 2021 ). Briefly, 10 µl of a 1 mg/ml antibody solution (scFvC-Hec1 rb , scFvC-Mad2-C rb , or Hec1 Fab 647 ) were placed directly atop growing HeLa cells in a 35 mm glass-bottomed dish.…”

Visualizing looping of two endogenous genomic loci using synthetic zinc‐finger proteins with anti‐FLAG and anti‐HA frankenbodies in living cells

“…Then, 4 μl of a mixture of plasmids (1 μg of smFLAG-KDM5B-24×MS2 and 0.5 μg of anti-FLAG FB-GFP) and purified MCP-HaloTag (130 ng) in PBS was pipetted on top of the cells after removing the Opti medium from the MatTek chamber, and ~106-μm glass beads (Sigma-Aldrich) were evenly distributed on top. The chamber was then tapped firmly seven times, and Opti medium was added back to the cells ( Cialek et al, 2021 ). After 3-hr bead loading, the cells were stained in 1 ml of 0.2 μM of JF646-HaloTag ligand ( Grimm et al, 2015 ) diluted in phenol-red-free complete DMEM.…”

Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells

“…Then 4 µL of a mixture of plasmids (1 µg of smFLAG-KDM5B-24×MS2 and 0.5 µg of anti-FLAG FB-GFP) and purified MCP-HaloTag (130 ng) in PBS was pipetted on top of the cells after removing the Opti-medium from the MatTek chamber, and ~106 µm glass beads (Sigma Aldrich) were evenly distributed on top. The chamber was then tapped firmly seven times, and Opti-medium was added back to the cells (Cialek et al, 2021). 3 h post bead loading, the cells were stained in 1 mL of 0.2 µM of JF646-HaloTag ligand (Grimm et al, 2015) diluted in phenol-red-free complete DMEM.…”

“…For puromycin treatments, U2OS cells seeded on 35mm MatTek chambers with 70% confluency were loaded with 1 µg of smFLAG-KDM5B-24×MS2 (Addgene #81084), 0.5 µg of anti-FLAG FB-GFP and 130 ng of purified MCP-HaloTag protein by bead loading (Cialek et al, 2021). 3 h post bead loading, imaging chambers were stained and washed as described above.…”