Astrocyte Isolation: A Method to Obtain Pure Preparation of Mouse Cortical Astrocytes
Published: April 30, 2023
Abstract
Source: Schildge, S. et al. Isolation and Culture of Mouse Cortical Astrocytes. J. Vis. Exp. (2013)
In this video, we describe the method to obtain a pure preparation of astrocytes from newly born mice.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Isolation and Plating of Mixed Cortical Cells Mixed cortical cell isolation for astrocyte cultures can be performed using P1 to P4 mouse pups. In order to achieve proper astrocyte density it is necessary to use 4 mouse pup cortices per T75 tissue culture flask. Therefore, volumes in the following protocol …
Representative Results
Figure 1. Dissection of postnatal (P3) mouse cortex. A) Whole brain. B) Brain after removal of olfactory bulbs and cerebellum. C) Isolation of cortices by peeling off the plate-like structure of the cortex from the brain. D, D') Cortex from ventral and dorsal site with meninges (black arrows indicate meningeal arteries). E) Cortex without meninges. Scale bar, 1.5 mm. <p…
Divulgaciones
The authors have nothing to disclose.
Materials
| Astrocyte culture media | |||
| DMEM high glucose | Life Technologies | 31966-021 | |
| FBS heat-inactivated | Life Technologies | 10082-147 | Final Concentration: 10% |
| Penicillin-Streptomycin | Life Technologies | 15140-122 | Final Concentration: 1% |
| Solution for brain tissue digestion | |||
| HBSS | Life Technologies | 14170-088 | |
| 2.5% Trypsin | Life Technologies | 15090-046 | Final Concentration: 0.25% |
| Otro | |||
| 70% (vol/vol) ethanol | Roth | 9065.2 | |
| Poly-D-Lysine | Millipor A-003 | A-003-E | |
| Water | PAA | S15-012 | Cell culture grade |
| PBS | PAA | H15-002 | Cell culture grade |
| 0.05% Trypsin-EDTA | Life Technologies | 25300-062 | |
| 0.45 µm Sterile filter | Sartorius | 16555 | |
| 3.5 cm petri dish | BD Falcon | 353001 | |
| 15 ml Falcon tube | BD Falcon | 352096 | |
| 50 ml Falcon tube | BD Falcon | 352070 | |
| 75 cm2 Tissue culture flask | BD Falcon | 353136 | |
| Forceps fine | Dumont | 2-1032; 2-1033 | #3c; #5 |
| Forceps flat tip | KLS Martin | 12-120-11 | |
| 13 cm surgical scissors | Aesculap | BC-140-R | |
| Stereomicroscope | Leica | MZ7.5 | |
| Stereomicroscope + Camera | Leica | MZ16F; DFC320 | |
| Microscope + Camera | Zeiss; Canon | Primo Vert; PowerShot A650 IS | |
| Centrifuge | Eppendorf | 5805000.017 | Centrifuge5804R |
| Orbital Shaker | Thermo Scientific | SHKE 4450-1CE | MaxQ 4450 |
| Water bath | Julabo | SW20; 37 °C |
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Astrocyte Isolation: A Method to Obtain Pure Preparation of Mouse Cortical Astrocytes. J. Vis. Exp. (Pending Publication), e20633, doi: (2023).