||Suspension (Some Adherent Cells)
||established from the mastocytoma tumor of a DBA/2 mouse treated with methylcolanthrene; used as target cells for cytotoxic T cell assays; as reported cells exhibit no effector activity in an antibody-dependent cell mediated cytotoxic system
||This cell line is a suitable transfection host.
||P815 cells phagocytose latex beads but not zymosan or BCG.
They do not function in antibody dependent cell mediated cytotoxicity.
Growth of the cells is not inhibited by dextran sulfate, LPS or PPD.
||Dulbecco's Modified Eagle's Medium (DMEM) modified to contain 2 mM L-glutamine, 4500 mg/L glucose and 10% of fetal bovine serum.
||Cultures can be maintained by addition or replacement of fresh medium.
Start cultures at 2 X 10e5 cells/mL and maintain between 1 X 10e5 and 1 X 10e6 cells/mL.
Adherent cells can be recovered by scraping.
NOTE: Maximum cell density at > 1 x 10e6 cells/ml
Population Doubling Time: 18-22 hrs
|Subculturing Medium Renewal
||Every 2 to 3 days
||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).
||Ralph P, et al. Lysozyme synthesis by established human and murine histiocytic lymphoma cell lines. J. Exp. Med. 143: 1528-1533, 1976. PubMed: 1083890
Ralph P, Nakoinz I. Antibody-dependent killing of erythrocyte and tumor targets by macrophage-related cell lines: enhancement by PPD and LPS. J. Immunol. 119: 950-954, 1977. PubMed: 894031
Ralph P, Nakoinz I. Direct toxic effects of immunopotentiators on monocytic myelomonocytic, and histiocytic or macrophage tumor cells in culture. Cancer Res. 37: 546-550, 1977. PubMed: 318922
Ralph P, Nakoinz I. Lipopolysaccharides inhibit lymphosarcoma cells of bone marrow origin. Nature 249: 49-51, 1974. PubMed: 4208429
Ralph P, et al. Lymphosarcoma cell growth is selectively inhibited by B lymphocyte mitogens: LPS, dextran sulfate and PPD. Biochem. Biophys. Res. Commun. 61: 1268-1275, 1974. PubMed: 4616699
Lundak RL, Raidt DJ. Cellular immune response against tumor cells. I. In vitro immunization of allogeneic and syngeneic mouse spleen cell suspensions against DBA mastocytoma cells. Cell. Immunol. 9: 60-66, 1973. PubMed: 4270287
Plaut M, et al. Studies on the mechanism of lymphocyte-mediated cytolysis. IV. Specificity of the histamine receptor on effector T cells. J. Immunol. 111: 389-394, 1973. PubMed: 4123976
Schmidt W, et al. Cell-free tumor antigen peptide-based cancer vaccines. Proc. Natl. Acad. Sci. USA 94: 3262-3267, 1997. PubMed: 9096381
Gonzalez Armas JC, et al. DNA immunization confers protection against murine cytomegalovirus infection. J. Virol. 70: 7921-7928, 1996. PubMed: 8892915
||Dr. T. kipnis, Universidade de São Paulo.