Single Cell Electroporation in Organotypic Brain Slice Cultures: An In Vitro Technique to Deliver Plasmids Inside Targeted Neurons in Brain Hippocampal Slices

Samenvatting

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

1. Prepare mouse organotypic hippocampal slice cultures using postnatal 6- to 7-day old mice of either sex.
   1. Prepare dissection media for organotypic slice culture consisting of (in mM): 238 sucrose, 2.5 KCl, 1 CaCl₂, 4 MgCl₂, 26 NaHCO₃, 1 NaH₂PO₄, and 11 glucose in deionized water, then gas with 5% CO₂/95% O₂ to a pH of 7.4.
   2. Prepare organotypic....

Resultaten

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.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
<strong>Plasmid preparation</strong>
Plasmid Purification Kit	Qiagen	12362
<strong>Organotypic slice culture preparation</strong>
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
<strong>Electrode preparation</strong>
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 &amp; 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