Bead Loading to Introduce Nucleic Acids Into Adherent Cultured Cells: A Technique to Load Fluorescent Protein-Encoding Plasmids Into Adherent Mammalian Cells

Begin with a culture of adherent mammalian cells in a culture plate. Transfer sterilized alkali-treated, micron-sized glass beads into the chamber of a customized bead loader apparatus. Cover the chamber opening using a mesh with optimum-size openings to allow the beads to pass through during loading. Secure the mesh with an imaging chamber assembly.

Remove media from the plate. Pipette a suspension of fluorescent reporter protein-encoding plasmid DNA into the plate for even distribution over the cells. Using the bead loader apparatus, gently disperse a monolayer of glass beads onto the cells. Tap the culture plate briefly with suitable force.

The glass beads briefly roll on top of the adherent cells, while the alkali treatment makes them less adherent to the cells. The impact of collisions between beads and cells transmits adequate strain creating localized disruptions in the cellular membranes. These formed transient pores of small dimensions facilitate the uptake of plasmid DNA into the cells while preventing the entry of large beads.

During the recovery period, the cellular membrane reseals, restoring the membrane integrity and entrapping the plasmid DNA. Add media to the plate and carefully aspirate any visible floating beads from the plate. Incubate the plate.

Successfully transfected cells containing plasmid DNA express the fluorescent reporter proteins, which can be visualized under a fluorescence microscope.

Remove the medium from the cells, and gently aspirate all medium from around the edges of the chamber. Then, tilt the chamber at approximately a 45-degree angle, and remove the remaining drop of media in the central microwell. Pipette the bead loading solution gently onto the glass microwell present in the center of the chamber.

Use the bead loading apparatus to gently disperse a monolayer of glass beads on top of the cells. Ensure that the beads cover the cells completely. Pinch the chamber with two fingers. Lift it around two inches, and bring it down firmly using a force approximately equivalent to dropping the dish from that height.

Gently add the medium back into the chamber by pipetting slowly onto the plastic side of the chamber. Aspirate any floating beads without disturbing the cells. The entire bead loading procedure should be performed relatively quickly, so the cells do not dry out when they are without medium. Then, incubate the cells for 0.5 to 2 hours in the incubator.