An efficient cell culture system for the studies of heterogeneous astrocytes: Time gradient digestion.
Aldh1l1
Astrocyte
Brain cortex
Gfap
Journal
Journal of neuroscience methods
ISSN: 1872-678X
Titre abrégé: J Neurosci Methods
Pays: Netherlands
ID NLM: 7905558
Informations de publication
Date de publication:
01 10 2021
01 10 2021
Historique:
received:
21
04
2021
revised:
27
06
2021
accepted:
16
07
2021
pubmed:
25
7
2021
medline:
6
10
2021
entrez:
24
7
2021
Statut:
ppublish
Résumé
Astrocytes are the most abundant glial cell type in mammal brain, but there exists a lot of unknown in cell development and cell function. We aim to establish an astrocytes culture system for obtaining highly enriched primary astrocytes from the neonatal mouse brain and separating Aldh1l1 C57BL/J6 mouse pups at postnatal 1-4 days were used for cell preparation. Brain cortex was collected and digested with 0.25% trypsin followed by 0.5 mg/ml DNase. Cells were plated on PDL-coated flasks. After 8-10 days culture, cells were shaken at 260 rpm for 4 h at 37 ℃ to remove oligodendrocytes and microglia cells. Time gradient digestion was performed to obtain astrocyte subtypes. The digestion time was 0-2 min and 2-4 min, and 4-6 min. Flow cytometry, Immunostaining, CCK-8 assay and EdU staining was carried out to investigate the purity of the astrocytes, the ability of cell proliferation and to identify different subtypes. After shaking, percentage of oligodendrocytes significantly decreased from 22.6 ± 3.6% to 7.4 ± 1.4% (CNPase Aldh1l1 A new astrocytes culture system with time gradient digestion was established. Highly enriched primary astrocytes from the neonatal mouse brain were obtained with short shaking time. Aldh1l1
Sections du résumé
BACKGROUND
Astrocytes are the most abundant glial cell type in mammal brain, but there exists a lot of unknown in cell development and cell function. We aim to establish an astrocytes culture system for obtaining highly enriched primary astrocytes from the neonatal mouse brain and separating Aldh1l1
NEW METHOD
C57BL/J6 mouse pups at postnatal 1-4 days were used for cell preparation. Brain cortex was collected and digested with 0.25% trypsin followed by 0.5 mg/ml DNase. Cells were plated on PDL-coated flasks. After 8-10 days culture, cells were shaken at 260 rpm for 4 h at 37 ℃ to remove oligodendrocytes and microglia cells. Time gradient digestion was performed to obtain astrocyte subtypes. The digestion time was 0-2 min and 2-4 min, and 4-6 min. Flow cytometry, Immunostaining, CCK-8 assay and EdU staining was carried out to investigate the purity of the astrocytes, the ability of cell proliferation and to identify different subtypes.
RESULTS
After shaking, percentage of oligodendrocytes significantly decreased from 22.6 ± 3.6% to 7.4 ± 1.4% (CNPase
COMPARISON WITH EXISTING METHODS
Aldh1l1
CONCLUSIONS
A new astrocytes culture system with time gradient digestion was established. Highly enriched primary astrocytes from the neonatal mouse brain were obtained with short shaking time. Aldh1l1
Identifiants
pubmed: 34302861
pii: S0165-0270(21)00227-2
doi: 10.1016/j.jneumeth.2021.109292
pii:
doi:
Substances chimiques
Glial Fibrillary Acidic Protein
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
109292Informations de copyright
Copyright © 2021 Elsevier B.V. All rights reserved.