| Derivation |
The CTX TNA2 cell line was established from primary cultures of type 1 astrocytes from brain frontal cortex tissue of 1 day old rats.
The cultures were transfected 3 days after initial plating with a DNA construct containing the oncogenic early region of SV40 under the transcriptional control of the human GFAP promoter (pGFA-SV-Tt) and pPGK-neo which contains the murine phosphoglycerate kinase gene promoter.
The transfectants were selected with G418 and cloned. |
| Comments |
The cells retain characteristics consistent with the phenotype of type 1 astrocytes.
About 20% of the cells have glial fibrillary acidic protein (GFAP) immunoreactivity.
The cells have a high affinity uptake mechanism for gamma aminobutyric acid (GABA) that is inhibitable by beta alanine.
The cells produce alpha 2 macroglobulin in amounts similar to those found in primary astrocytes but produce transferrin in much lesser amounts.
This line does not produce proenkephalin A, does not express the O4 or A2B5 epitopes characteristic of type 2 astrocytes, and does not express galactocerebroside.
SV40 T-antigen was found in the nuclei of over 95% of the cells examined by immunostaining. |
| Subculturing |
Volumes used in this protocol are for 75 cm2 flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
- Remove and discard culture medium.
- Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.
- Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask Cells may be placed at 37°C to facilitate dispersal (usually within 5 to 15 minutes). Observe cells under an inverted microscope until cell layer is dispersed.
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. The cells will come off in sheets and are difficult to dissociate.
- Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
- Add appropriate aliquots of the cell suspension to new culture vessels.
- Incubate cultures at 37°C.
Subcultivation Ratio: A subcultivation ratio of 1:2 to 1:8 is recommended
Medium Renewal: Every 2 to 3 days
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 10 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 3rd edition, published by Alan R. Liss, N.Y., 1994. |
| References |
Radany EH, et al. Directed establishment of rat brain cell lines with the phenotypic characteristics of type 1 astrocytes. Proc. Natl. Acad. Sci. USA 89: 6467-6471, 1992. PubMed: 1378628
Hay, R. J., Caputo, J. L., and Macy, M. L., Eds. (1992), ATCC Quality Control Methods for Cell Lines. 2nd edition, Published by ATCC.
Caputo, J. L., Biosafety procedures in cell culture. J. Tissue Culture Methods 11:223-227, 1988.
Fleming, D.O., Richardson, J. H., Tulis, J.J. and Vesley, D., (1995) Laboratory Safety: Principles and Practice. Second edition, ASM press, Washington, DC.
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