Astrocyte Isolation: A Method to Obtain Pure Preparation of Mouse Cortical Astrocytes

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…

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%
기타
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