|Cell Line:||WEHI 164|
Vulgar Name: Mouse; Balb/C
|Derivation:||This line was originally established by M. Rollinghoff and N.L. Warner from a fibrosarcoma induced by subcutaneous injections of 3-methylcholanthrene.|
|Applications:||These cells can be used as target cells for short term chromium release cytotoxicity assays.|
|Additional info:||After pretreatment with actinomycin D, the cells are highly sensitive to human cytotoxic monocytes, to human tumor necrosis factor and to lymphotoxin.|
|Culture Medium:||RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate and 10% of fetal bovine serum.|
Remove medium, add fresh 0.25% trypsin, 0.02% EDTA solution for 1 to 2 minutes.
Remove trypsin and place the flask at 37°C until the cells detach (about 3 to 5 minutes).
Add fresh medium, aspirate and dispense into new flasks.
NOTE: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 12 in Culture of Animal Cells, a manual of Basic Technique by R. Ian Freshney, 6th edition, published by Alan R. Liss, N.Y., 2010.
Medium Renewal: Every 2 to 3 days.
Subcultivation ratio: 1:5 to 1:20
|Culture Conditions:||Atmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37°C|
|Cryopreservation:||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).|
|References:||Ziegler-Heitbrock HW, Riethmuller G. A rapid assay for cytotoxicity of unstimulated human monocytes. J. Natl. Cancer Inst. 72: 23-29, 1984. PubMed: 6582301 Ziegler-Heitbrock HW, et al. Killer cell activity of human monoblastic leukemia cells as detected with a monocyte-specific target cell. Blood 65: 8-14, 1985. PubMed: 3155490 Sugarman BJ, et al. Recombinant human tumor necrosis factor-alpha: effects on proliferation of normal and transformed cells in vitro. Science 230: 943-945, 1985. PubMed: 3933111 Rollinghoff M, Warner NL. Specificity of in vivo tumor rejection assessed by mixing immune spleen cells with target and unrelated tumor cells. Proc. Soc. Exp. Biol. Med. 144: 813-818, 1973. PubMed: 4543620 Knowlton KU, et al. A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the woodruff variant of coxsackievirus B3. J. Virol. 70: 7811-7818, 1996. PubMed: 8892902|
|Depositors:||Pedro Paulo Elsas; Universidade Federal do Rio de Janeiro.|