Optimized and efficient preparation of astrocyte cultures from rat spinal cord

Abstract

Astrocytes constitute a major class of glial cells in the CNS, and play crucial roles in physiological functioning, performance and maintenance of the CNS, as well as promotion of neuronal migration and maturation. Astrocytes have also been directly and indirectly implicated in the pathophysiology of various trauma occurrences, development of neurodegenerative diseases and nerve regeneration. To further understand mechanisms by which astrocytes elicit these effects, the first critical step in the study of astrocytes is the preparation of purified astrocytes cultures. Here we describe a simple and convenient procedure for producing rat primary astrocyte cultures of high purity, viability and proliferation. For astrocyte culture, we have optimized the isolation procedures and cultivation conditions including coating substrates, enzyme digestion, seeding density and composition of the culture medium. Using immunofluorescent antibodies against GFAP and OX-42 in combination of Hoechst 33342 fluorescent staining, we found that the purity of the astrocyte cultures was >99%. Astrocytes had high viability as measured by 3-(4, 5-dimethyl-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. In addition, flow cytometric analysis was used to measure and observe variations in the cell cycle after 1-2 passages and proliferation of astrocytes was detected with a high percentage of cells stand in S+G 2 /M phase. Therefore, the method described here is ideal for experiments, which require highly pure astrocyte cultures.

De-differentiation Response of Cultured Astrocytes to Injury Induced by Scratch or Conditioned Culture Medium of Scratch-Insulted Astrocytes

“…The cultures were grown in DMEM containing 20% FCS, supplemented with 2 mM glutamine, 25 mM HEPES, 50 U/ml penicillin, and 50 lg/ml streptomycin at 37°C with 5% CO 2 . About 48-60 h in vitro later, purification of cell was undertaken according to previously protocol (Yang et al 2006).…”

Evidence for Heterogeneity of Astrocyte De-Differentiation in vitro: Astrocytes Transform into Intermediate Precursor Cells Following Induction of ACM from Scratch-Insulted Astrocytes

“…Our interpretations of the results were given as follows: astrocytes had been cultured according to our previous study protocol (Yang et al 2006(Yang et al , 2009. And also these cells had been maintained and subcultured in culture medium containing serum over 20 days.…”

“…Primary astrocyte culture, purification, and wound model were undertaken and processed according to previously described by Yang et al (2006Yang et al ( , 2009 in accordance with guidelines at the animal Care and Use committee of China. In this study, the purity of the astrocyte cultures was never below 98% as confirmed by anti-glial fibrillary acidic protein (GFAP).…”

A Mini Review on My Study of the Spinal Cord Injury

“…It had no effect on her left leg, but her right leg swung a few times and stopped. It clearly proved that her right leg did have a mild ability to move [4][5][6][7].…”