B35

B35 cells can be stimulated to differentiate in the presence of dibutyryl cyclic AMP (cAMP) or by serum deprivation.

The cells also may be used to study the metabolism and physiology of nervous tissue and the pathology of nervous disorders.

They are easily transfected with plasmid DNA.

Rats were inoculated with N-nitrosoethylurea (NEU) 15 days after conception. Tumors found in the central nervous system (CNS) 4 to 10 months after birth were excised, minced, adapted to culture and cloned. RefSchubert D, et al. Clonal cell lines from the rat central nervous system. Nature 249: 224-227, 1974. PubMed: 4151463

The cells retain glutamic acid decarboxylase (GAD) and choline acetyltransferase activities; express gamma aminobutyric acid (GABA). The cells are negative for S100 (S-100) protein. RefSchubert D, et al. Clonal cell lines from the rat central nervous system. Nature 249: 224-227, 1974. PubMed: 4151463 The cells are positive for neuron specific enolase. RefVinores SA, et al. Immunoradiometric and immunohistochemical demonstration of neuron-specific enolase in experimental rat gliomas. Cancer Res. 44: 2595-2599, 1984. PubMed: 6722796

A culture submitted to the ATCC in October 2002 was found to be contaminated with mycoplasma. Progeny were cured by a 21-day treatment with BM Cycline. The cells were assayed for mycoplasma, by the Hoechst stain, PCR and the standard culture test, after a six-week period following treatment. All tests were negative.

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 75 cm² tissue culture flask and dilute with the recommended complete culture medium (see the specific batch information for the culture recommended dilution ratio). 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).
4. 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 0.05% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
3. Add 2.0 to 3.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
   Note: To avoid clumping, do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by pipetting gently.
5. Add appropriate aliquots of cell suspension to new culture vessels.
6. Incubate culture vessels at 37°C.

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