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

Abstract

Source: Cialek, C. A. et al. Bead Loading Proteins and Nucleic Acids into Adherent Human Cells. J. Vis. Exp. (2021).

In this video, we describe the procedure to introduce nucleic acids into adherent mammalian cells using bead loading.

Protocol

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 ....

Results

Figure 1: Bead loading apparatus, technique, and timeline

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Materials

List of materials used in this article

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)