Mouse fibroblasts were cultured by three different procedures: (a) changing the 0.2 ml/cm2 of growth medium every 2nd d and seeding 1×105 cells/cm2 after confluency; (b) changing the 0.4 ml/cm2 of growth medium only at subculture performed at confluency by a 1∶2 split and keeping the bottles incubated on a rocking platform; (c) the same as Method b but keeping the bottles stationary throughout culture. By Method a no lines were immortalized over 36 experiments whereas Method b gave 1/4 immortalized lines and Method c gave 10∶12 immortalized lines. Cells always went into crisis at the 9th to 11th doubling. Immortalized lines had a tetraploid DNA content.
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This work was partially supported by CNR CT No. 82.00183.04. Judy Baggott, Felice and Vincenzo de Ceglie, Flavia Boniardi, and Antonella Bottazzi helped prepare this manuscript.
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Curatolo, L., Erba, E. & Morasca, L. Culture conditions induce the appearance of immortalized C3H mouse cell lines. In Vitro 20, 597–601 (1984). https://doi.org/10.1007/BF02619607
- culture procedure
- immortalized cell lines
- flow cytometry