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

Offenlegungen

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)

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Diesen Artikel zitieren
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).

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