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

Published: April 30, 2023

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 Follow instructions to install the software from https://github.com/bsbrl/Autoinjector. 2. Preparation of Reagents and Pipettes Agarose: Prepare 3% agarose by separately dissolving 3 g of wide range agarose and 3 g low-melting point…

Offenlegungen

The authors have nothing to disclose.

Materials

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

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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. J. Vis. Exp. (Pending Publication), e20989, doi: (2023).

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