||Single, round cells in suspension
||It can be used to quantify the presence of IL-3 in biological fluid.
||The DA-1 cell line is dependent upon IL-3 (interleukin-3).
||Iscove's modified Dulbecco's medium with 2mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate + 0,25mM mercaptoethanol + IL-3 (either 30% vol conditioned medium of WEHI-3B cell line or 20 U/mL mIL-3) + 5% FBS
||Cultures can be maintained by addition or replacement of fresh medium.
Maintain cultures from 0.2-0.4 x 10e6 cells/mL; cells grow fast.
By favoring selective growth, suboptimal culture of cytokine-dependent cell lines may promote outgrowth of factor-independent subclones. Selective conditions include cytokine insufficiency and inadequate cell density. Thus the BCRJ cannot guarantee indefinite stability of factor-dependence of cell lines.
Maximal density at 0.8 x 10e6 cells/mL.
||Atmosphere: air, 95%; carbon dioxide (CO2), 5%
||95% FBS + 5% DMSO (Dimethyl sulfoxide)
|Thawing Frozen Cells:
||SAFETY PRECAUTION: Is highly recommend that protective gloves and clothing always be used and a full face mask always be worn when handling frozen vials. It is important to note that
some vials leak when submersed in liquid nitrogen and will slowly fill with liquid nitrogen. Upon thawing, the conversion of the liquid nitrogen back to its gas phase may result in the vessel exploding or blowing off its cap with dangerous force creating flying debris.
1. Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the Oring 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. For cells that are sensitive to DMSO is recommended that the cryoprotective agent be removed immediately. Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 x g for 5 to 7 minutes.
4.Discard the supernatant and Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio).
5. Incubate the culture in a appropriate atmosphere and temperature (see "Culture Conditions" for this cell line).
NOTE: 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 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).
||Pierce, J. H., Di Fiore, P. P., Aaronson, S. A., Potter, M., Pumphrey, J., Scott, A., Ihle, J. N. (1985). Neoplastic transformation of mast cells by Abelson-MuLV: abrogation of IL-3 dependence by a nonautocrine mechanism. Cell 41 ( 3 ): 685-693. PMID: 2988783
||Marcelo Barciski, Universidade Federal do Rio de Janeiro.