Cells - B35
Cells derived from nitrosoethylurea (NEU) induced tumours in newborn rats (approx 5 neural, 10 glial, + others generated). These cells have small round neuronal morphology with extending, sometimes branched, neurites. May also have a bipolar appearance. They rapidly proliferate with 24 h doubling times.
SubCulturing: Cells from confluent cultures (approximately 20 million cells per 75 sq. cm.) are dislodged from the flask surface, aspirated and dispensed into new flasks. Trypsin - EDTA dissociation can be used, but it is better to do so infrequently. Cultures should be maintained at medium density. Seed new flasks at a density of at least 5 X 106 viable cells per 75 sq. cm. flask.
Growth Properties: monolayer
Original cells from P. Jeffries with permission from David Schubert (1995). Rapidly proliferate with 24 h doubling times. Can induce differentiation by serum withdrawal and dbcAMP addition. Can be transfected using lipofectamine technique. Medium: Dulbecco's modified Eagle's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose and 1.0 mM sodium pyruvate, 90%; fetal bovine serum, 10% also with Pen/strep 1%.
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 [PubMed: 4151463]. B35 cells can be stimulated to differentiate in the presence of dibutyryl cyclic AMP (cAMP) or by serum deprivation. They are easily transfected with plasmid DNA. 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 [PubMed: 4151463]. The cells are positive for neuron specific enolase [PubMed: 6722796].The cells also may be used to study the metabolism and physiology of nervous tissue and the pathology of nervous disorders. 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.
ATCC complete growth medium: The base medium for this cell line is ATCC-formulated Dulbecco's Modified Eagle's Medium, Catalog No. 30-2002. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%. Temperature: 37.0°C
Protocol: Subculture before confluency
- Remove and discard culture medium.
- Briefly rinse the cell layer with 0.05% (w/v) Trypsin- 0.53 mM 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 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.
- 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 1:5 to 1:8 is recommended
Medium Renewal: Every 2 to 3 days