Single Cell Electroporation in Organotypic Brain Slice Cultures: An In Vitro Technique to Deliver Plasmids Inside Targeted Neurons in Brain Hippocampal Slices
Published: April 30, 2023
Abstract
Source: Keener, D. G. et al. Single-Cell Electroporation Across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons. J. Vis. Exp. (2020)
This video demonstrates the protocol for single-cell electroporation of genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice cultures. The technique is useful to examine specific molecular and physiological functions of neurons, including cell autonomous mechanisms and transsynaptic protein interactions.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Slice Culture Preparation Prepare mouse organotypic hippocampal slice cultures using postnatal 6- to 7-day old mice of either sex. Prepare dissection media for organotypic slice culture consisting of (in mM): 238 sucrose, 2.5 KCl, 1 CaCl2, 4 MgCl2, 26 NaHCO3, …
Representative Results
Figure 1: Two representative glass pipette images. (A) Display lower resistance (6.5 MΩ) pipettes, used in this protocol, and (B) higher resistance (10.4 MΩ) pipettes typical for electroporation protocols. Fig…
Açıklamalar
The authors have nothing to disclose.
Materials
| Plasmid preparation | |||
| Plasmid Purification Kit | Qiagen | 12362 | |
| Organotypic slice culture preparation | |||
| 6-well plates | GREINER BIO-ONE | 657160 | |
| Dumont #5/45 Forceps | FST | #5/45 | Angled dissection forceps for organotypic slice culture preparation |
| Flask Filter Unit | Millipore | SCHVU02RE | Filtration and storage of culture media |
| Incubator | Binder | BD C150-UL | |
| McIlwain Tissue Chopper | TED PELLA, INC. | 10180 | Tissue chopper for organotypic slice culture preparation |
| Millicell Cell Culture Insert, 30 mm | Millipore | PIHP03050 | Organotypic slice culture inserts |
| Osmometer | Precision Systems | OSMETTE II | |
| PTFE coated spatulas | Cole-Parmer | SK-06369-11 | |
| Scissors | FST | 14958-09 | |
| Stereo Microscope | Olympus | SZ61 | |
| Sterile Vacuum Filtration System | Millipore | SCGPT01RE | Filtration and storage of aCSF |
| Electrode preparation | |||
| Capillary Glasses | Warner Instruments | 640796 | |
| Micropipetter Puller | Sutter Instrument | P-1000 | Puller |
| Oven | Binder | BD (E2) | |
| Puller Filament | Sutter Instrument | FB330B | Puller |
| 3.5 mm Falcon Petri Dishes | BD Falcon | 353001 | |
| Airtable | TMC | 63-7512E | |
| CCD camera | Q Imaging | Retiga-2000DC | Camera |
| Electroporation System | Molecular Devices | Axoporator 800A | Electroporator |
| Fluorescence Illumination System | Prior | Lumen 200 | |
| Manipulator | Sutter Instrument | MPC-385 | Manipulator |
| Metamorph software | Molecular Devices | Image acquisition | |
| Peristaltic Pump | Rainin | Dynamax, RP-2 | Perfusion pump |
| Shifting Table | Luigs & Neuman | 240 XY | |
| Speaker | Unknown | Speakers connected to the electroporator | |
| Stereo Microscope | Olympus | SZ30 | |
| Table Top Incubator | Thermo Scientific | MIDI 40 | |
| Upright Microscope | Olympus | BX61WI |
Etiketler
Bu Makaleden Alıntı Yapın
Single Cell Electroporation in Organotypic Brain Slice Cultures: An In Vitro Technique to Deliver Plasmids Inside Targeted Neurons in Brain Hippocampal Slices. J. Vis. Exp. (Pending Publication), e20697, doi: (2023).