Visualization of Calcium Influx in Ventral Midbrain Neurons Derived from Mouse Embryos

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 demonstrates the real-time visualization of calcium influx in ventral midbrain neurons derived from mouse embryos. Using a viral vector to express a calcium indicator, the neurons are imaged under a confocal microscope to track changes in fluorescence intensity during spontaneous and neurotransmitter-induced calcium activity.

Protocol

1. Plating the cells

NOTE: Based on experience, about 100,000 viable cells per embryo are collected. 2–3-month-old timed pregnant mice typically have litter sizes of 8-10 embryos; therefore, a rough estimate for total yield of cells per timed pregnant mouse is approximately 1 million cells.

1. Using a hemocytometer, perform a cell count and then dilute the suspension to 2,000 cells/µL using cell culture medium. Triturate briefly to mix.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35 mm uncoated plastic cell culture dishes	VWR	25382-348
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
Hanks-balanced Salt Solution (HBSS) 1x	ThermoFisher	14175095	500 mL
HEPES	VWR	101170-478
HeraCell 150 CO₂ incubator	Heraeus (ThermoFisher)
Laminin	Sigma-Aldrich	L2020
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
pAAV.Syn.GCaMP6f.WPRE.SV40	Addgene	100837-AAV1	Titer: 1.00E+13 gc/ml
Potassium Chloride (KCl), anhydrous	Sigma-Aldrich	746436
Pump Head Tubing Pieces For MPII	Harvard Apparatus	55-4148
Sodium Chloride (NaCl), anhydrous	Sigma-Aldrich	746398
Time-pregnant female C57BL/6 mice	Texas A&M Institue for Genomic Medicine