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.

Evaluating Riboglow-FLIM probes for RNA sensing

“…This observation is consistent with previous work on Riboglow 14,16,17 and other studies in which bead loading was utilized to introduce proteins into live cells, and confirms that this process is not impactful to cell health. 27,28…”

Spontaneous Confinement of mRNA Molecules at Biomolecular Condensate Boundaries

“…The cells were treated with 500 μM sodium arsenate to induce oxidative stress and form SGs [ 9 , 22 ]. We enzymatically synthesized a 1600 nt long, 5′-capped, poly-adenylated, and Cy5-labeled mRNA [ 16 ] and introduced it into both cell types via bead loading [ 23 ]. The cells were then placed onto a glass bottom cell dish and incubated for 45 min in the case of UGG cells and for 60 min in the case of UGD cells.…”

“…Messenger RNA was loaded into cells via bead loading [ 16 ]. Briefly, the medium was removed, and the cells were washed twice with 1 mL 1× PBS buffer; then, 5 µL of RNA solution in 1× PBS buffer (200 ng) was added to the center of the glass dish, followed by an addition of glass beads.…”

Single molecule imaging of the central dogma reveals myosin-2A gene expression is regulated by contextual translational buffering

“…Translation sites in fixed cells were labeled anti-Flag labeling (FUJIFILM, Wako) followed by Cy3 or Alexa 594 conjugated anti-mouse secondary antibodies (Jackson Immunoresearch and Invitrogen). In live cells, translation sites were labeled by bead loading Flag-Cy3 Fab antibody fragments as previously described 36 . Cy3-Flag antibodies detect nascent Flag-MYH9 proteins where both mCherry and mClover signals are absent due to the delay in fluorescence protein maturation.…”