Bead Loading to Introduce Nucleic Acids Into Adherent Cultured Cells: A Technique to Load Fluorescent Protein-Encoding Plasmids Into Adherent Mammalian Cells
Published: April 30, 2023
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. 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
Divulgations
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) |
Tags
Citer Cet Article
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).