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

Protocolo

1. Bead loading cells

NOTE: If required, wash the cells briefly with phosphate-buffered saline (PBS) and then add 2 mL of the optimal medium. Incubate for at least 30 min.

1. Make a solution of 3-8 µL containing the desired plasmids, protein, and/or particles. Use ~1 µg (0.1-1 pmol) of each type of plasmid and ~0.5 µg (0.01 nmol) of protein, depending on experimental requirements. Use a low-retention tube for proteins so that they are not left behind on the tube walls. Bring the solution up to a minimum of 3 µL with PBS, and adjust the solution volume to coat the entire area of cells to be loaded (i.e., the chamber's microwell, Figure 1B).
2. Mix the solution thoroughly by pipetting up and down and/or flicking the tube. Briefly spin the solution down to the bottom of the tube in a tabletop microfuge.
3. Transfer the bead loading solution and the chamber of cells into a tissue culture hood. Perform the remaining steps in the tissue culture hood using sterile technique.
4. Remove the medium from the cells and temporarily store it in a sterile tube. Gently aspirate all medium from around the edges of the chamber and tilt the chamber at approximately a 45° angle and remove the remaining drop of media in the center microwell. During medium removal, make sure to avoid letting the pipette tip touch the glass, which may result in cell peeling and loss. Move quickly to the next step so that the cells are not dry for long.
5. Gently pipette the bead loading solution onto the glass microwell in the center of the chamber. Optional: Incubate with gentle rocking for ~30 s without allowing the chamber to dry up completely.
6. Gently disperse a monolayer of glass beads on top of the cells, preferably using a bead loading apparatus (Figure 1A). Ensure that the beads cover the cells in the glass-bottom microwell completely.
7. Pinching the chamber with two fingers, tap it against the hood surface by lifting it ~2 inches and bringing it down firmly. Use a force approximately equivalent to dropping the dish from that height. Repeat for a total of ~10 taps.
   NOTE: Ensure that the taps do not substantially peel the cells. Tapping can be optimized for the cell type. If cells load poorly, tap harder; however, if many cells peel off, tap more lightly.
8. Gently add medium back into the chamber by pipetting slowly onto the plastic side of the chamber. Try to aspirate any floating beads without disturbing the cells. Add more pre-warmed media at this step if too much was removed. Incubate the cells for 0.5-2 h in the incubator.

Resultados

Figure 1: Bead loading apparatus, technique, and timeline

Materiais

Name	Company	Catalog Number	Comments
10 cm cell culture dishes	VWR	82050-916	Use to culture cells
35 mm cell culture dishes	Falcon	353001	Use to construct bead loader
Attofluor Cell Chamber	Thermo Fisher Scientific	A7816	Use to construct the custom bead loader
DMEM, high glucose, no glutamine	Thermo Fisher Scientific	11960069	Use in general cell culture
Glass bottom dishes, 35 mm, #1.5, 14 mm glass	MatTek Corporation	P35G-1.5-14-C	Seed cells onto these chambers for imaging
Glass beads, acid washed, ≤106 µm	Millipore Sigma	G4649	Sprinkle on cells to bead load plasmid DNA and proteins
Phosphate Buffered Saline (PBS)	Thermo Fisher Scientific	AM9625	Working stock of sterile 1X PBS
Phenol-free DMEM	Thermo Fisher Scientific	31053036	Use on cells before imaging
Opti-MEM, Reduced Serum Medium	Thermo Fisher Scientific	31985070	Optimal media for incubating cells before bead loading (optional step)