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…
Declarações
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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Citar este artigo
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).