CTX TNA2

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.

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.

1. Check all containers for leakage or breakage.
2. Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below ­-130°C, preferably in liquid nitrogen vapor, until ready for use.

1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water.  Thawing should be rapid (approximately 2 minutes).
2. Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
3. Transfer the vial contents to a centrifuge tube containing  9.0 mL complete growth medium and spin at approximately 125 x g for 5 to 7 minutes.
4. Resuspend cell pellet with the recommended complete growth medium (see the specific batch information for the culture recommended dilution ratio). and dispense into a 25 cm² or a 75 cm² culture flask.  It is important to avoid excessive alkalinity of the medium during recovery of the cells.  It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
5. Incubate the culture at 37°C in a suitable incubator.  A 5% CO₂ in air atmosphere is recommended if using the medium described on this product sheet.

1. Remove and discard culture medium.
2. 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.
3. 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.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.

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.

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