Image-Guided Robotic Cell Microinjection: An Automated High-Throughput Technique for the Precise Delivery of Fluorescently-Labeled Polysaccharides Into Single Neuronal Cells in Organotypic Brain Slice Cultures

Abstract

Source: Shull, G. et al. Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection. J. Vis. Exp. (2021)

In this video, we demonstrate the robotic microinjection of fluorescently-labeled polysaccharides into pyramidal neurons in organotypic slices of mouse embryonic brain using an autoinjector. The autoinjector is a robotic platform that utilizes images acquired from a microscope for the precise delivery of biomolecules of interest into single cells.

Protocol

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

1. Installation of Software

1. Follow instructions to install the software from https://github.com/bsbrl/Autoinjector.

2. Preparation of Reagents and Pipettes

1. Agarose: Prepare 3% agarose by separately dissolving 3 g of wide range agarose and 3 g low-melting point agarose in 100 mL of cell culture-grade PBS in two separate 200 mL glass bottles, respectively. Store at room temperature for up to 3 months.
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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals
Agarose, Low Melt	Carl Roth	Cat# 6351.2
Agarose, Wild Range	Sigma-Aldrich	Cat# A2790
Best-CA 221 Glue	Best Klebstoffe GmbH & Co.KG	Cat# CA221-10ml
B-27 Supplement	Thermo Fisher Scientific	Cat# 17504044
Distilled Water
DMEM-F12, CO2 independent (w/o Phenol red)	Sigma-Aldrich	Cat# D2906
DMEM-F12, CO2 independent (with Phenol red)	Sigma-Aldrich	Cat# D8900
HEPES-NAOH, pH 7.2, 1M (HEPES buffer)	Carl Roth	Cat# 9105.3
L-Glutamine, 200 mM	Thermo Fisher Sientific	Cat# 25030024
Mowiol 4-88	Sigma-Aldrich	Cat# 81381
N-2 Supplement	Thermo Fisher Scientific	Cat# 17502048
Neurobasal Medium	Thermo Fisher Scientific	Cat# 21103049
Nuclease-free water	Thermo Fisher Scientific	Cat# AM9937
O2 (40%), CO2 (5%), N2 (55%) Mix, 50 liters
PBS
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	Cat# 15140122
Rat serum	Charles River Laboratories
Sodium bicarbonate (NaHCO3)	Merck	Cat# 106323
Sodium hydroxide (NaOH)	Merck	Cat# 106482
Tyrode’s salt	Sigma	Cat# T2145-10x1L)
Equipment
Borosilicate glass capillaries, 1.2 mm outer diameter x 0.94 mm inner diameter	Sutter Instruments	Cat# BF-120-94-10
Bottle-top filter system, 500 mL	Corning	Cat# 430769
Computer PC
Custom pressure rig		Custom pressure rig
Electronic pressure regulator	Parker Hannifin	Cat# 990-005101-002
Falcon tubes, 15 mL	Corning	Cat# 430791
Falcon tubes, 50 mL	Corning	Cat# 430829
Flaming/ Brown micropipette puller	Sutter Instruments	Cat# P-97
Forceps, Dumont no. 3	Fine Science Tools	Cat# 11231-30
Forceps, Dumont no. 5	Fine Science Tools	Cat# 11255-20
Forceps, Dumont no. 55	Fine Science Tools	Cat# 11252-20
Heating block	Labtech International	Cat # Dri block Digi2
Inverted fluorescence microscope	Zeiss	Cat# Axiovert 200
Light source	Olympus	Cat# Highlight 3100
Manual pressure regulator	McMaster Carr	Cat# 0-60 PSI 41795K3
Microloader Tips	Eppendorf	Cat# 5242956.003
Microcontroller	Arduino	Cat# Arduino Due
Microscope camera Hamamatsu Orca Flash 4.0 V3
Motorized stage XY for microscope
Pasteur pipettes, plastic
Petri dish, 60 x 15 mm	Greiner	Cat# 628102
Petri dish, 35 x 10 mm	Nunc	Cat# 153066
Petri dish, 34 x 14 mm, including Microwell no. 1.5 cover glass	MatTek	Cat# P35G-1.5-14-C
Pipette holder	Warner Instruments	Cat# 64-2354 MP-s12u
Pipette and tips
Puller filament, 3.0-mm square box filament	Sutter Instrument	Cat# FB330B
Slice culture incubation box	MPI-CBG	Cat# custom made
Solenoid valve	Cat# LHDA053321H-A
Tabletop centrifuge	Heraeus	Cat# 5431622
Thermometer
Three-axis Manipulator	Sensapex Inc	Cat# tree-axis uMP
Vibratome	Leica	Cat# VT1000s
Whole-embryo-culture-system incubator	Ikemoto Company	Cat# RKI-10-0310
Waterbath
Software and Algorithms
Arduino	Arduino
Fiji	RRID: SCR_002285
Python	Python Software foundation	Python 2.7.12
ZEN		RRID: SCR_013672