Bead Loading to Introduce Nucleic Acids Into Adherent Cultured Cells: A Technique to Load Fluorescent Protein-Encoding Plasmids Into Adherent Mammalian Cells
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. 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 n…
Representative Results
Figure 1: Bead loading apparatus, technique, and timeline
Divulgazioni
The authors have nothing to disclose.
Materials
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)
Bead Loading to Introduce Nucleic Acids Into Adherent Cultured Cells: A Technique to Load Fluorescent Protein-Encoding Plasmids Into Adherent Mammalian Cells. J. Vis. Exp. (Pending Publication), e20985, doi: (2023).