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).

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