Infecting Primary Mouse Midbrain Neurons with Engineered Adeno-Associated Viruses

Abstract

Source: Bancroft, E. A., et al., Quantifying Spontaneous Ca²⁺ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons. J. Vis. Exp. (2020).

This video shows infecting primary mouse midbrain neurons with Adeno-Associated Viruses carrying a fluorescent calcium indicator. The indicator's fluorescence confirms successful infection and enables real-time monitoring of neuronal infection.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

NOTE: Cell culture solutions should be prepared using sterile procedures in a biological safety cabinet and filtered at 0.2 µm to prevent contamination.

1. Preparation of solutions and culture medium

1. Prepare laminin coating solution by diluting 20 µL of 1 mg/mL laminin stock into 2 mL of sterile distilled H₂O. Prepare on the day of dissection.
2. Prepare 10% equine (horse) serum (ES) stop solution ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10X NA 0.3 water-immersion objective	Olympus	UMPLFLN10XW
12 mm circular cover glass No. 1	Phenix Research Products	MS20-121
20X NA 0.85 oil-immersion objective	Olympus	UPLSAPO20XO
35 mm uncoated plastic cell culture dishes	VWR	25382-348
40X NA 0.3 water-immersion objective	Olympus	LUMPLFLN40XW
60X NA 1.35 oil-immersion objective	Olympus	UPLSAPO60XO
Ampicillin (sodium)	Gold Bio	A-301-25
B-27 supplement	ThermoFisher	17504044	50x stock
Binocular Microscope	Kent Scientific	KSCXTS-1121
Bovine serum albumin (BSA)	Sigma-Aldrich	A7030
Calcium Chloride (CaCl2), anhydrous	Sigma-Aldrich	746495
Deoxyribonuclease I (DNase)	Sigma-Aldrich	DN25
D-glucose, andydrous	Sigma-Aldrich	RDD016
DMEM + GlutaMAX medium	ThermoFisher	10569010	500 mL
Equine serum	ThermoFisher	26050088	heat-inactivated
Fiber Optic Illuminator, 100V	Kent Scientific	KSC5410
Filter System, PES 22UM 250ML	VWR	28199-764
Fluoview 1000 confocal microscope	Olympus
Fluoview 1200 confocal microscope	Olympus
GlutaMAX supplement	ThermoFisher	35050061
Hanks-balanced Salt Solution (HBSS) 1x	ThermoFisher	14175095	500 mL
HEPES	VWR	101170-478
HeraCell 150 CO2 incubator	Heraeus (ThermoFisher)
ImageJ v1.52e	NIH
IRIS-Fine Scissors (Round Type)-S/S Str/31*8mm/13cm	RWD	S12014-13
Kanamycin monosulfate	Gold Bio	K-120-25
Laminin	Sigma-Aldrich	L2020
L-Ascorbic acid	Sigma-Aldrich	A7506
L-glutamic acid	VWR	97061-634
Magnesium Chloride (MgCl2), andydrous	Sigma-Aldrich	M8266
MPII Mini-Peristaltic Pump, 115/230 VAC, 50/60 Hz	Harvard Apparatus	70-2027
MULLER Micro Forceps-Str, 0.15mm Tips, 11cm	RWD	F11014-11
NBQX	Hello Bio	HB0443
Neurobasal medium	ThermoFisher	21103049	500 mL
Normal goat serum (NGS)	Abcam	ab7481
pAAV.Syn.GCaMP6f.WPRE.SV40	Addgene	100837-AAV1	Titer: 1.00E+13 gc/ml
Papain	Worthington Biomedical Corporation	LS003126
Penicillin streptomycin	ThermoFisher	15140122	10,000 U/mL
Phosphate-buffered saline (PBS) 1x	ThermoFisher	10010049	500 mL
Poly-L-lysine	Sigma-Aldrich	P4832
Poly-L-ornithine	Sigma-Aldrich	P4957
Potassium Chloride (KCl), anhydrous	Sigma-Aldrich	746436
Pump Head Tubing Pieces For MPII	Harvard Apparatus	55-4148
Sodium Chloride (NaCl), anhydrous	Sigma-Aldrich	746398
Sucrose	Sigma-Aldrich	S7903	BioXtra, ≥99.5% (GC)
Time-pregnant female C57BL/6 mice	Texas A&M Institue for Genomic Medicine
Triton X-100	Sigma-Aldrich	X100	500 mL
Wide-bore blue pipette tips P1000	VWR	83007-380