By James A. Spudich (Eds.)
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Extra info for Dictyostelium discoideum: Molecular Approaches to Cell Biology
The pan is sealed as usual with Saran wrap. We also occasionally use a 9-hole version. E. Use of Filters in Labeling Experiments As noted above, this can be done at any stage and for any period simply by switching filters to fresh pads. However, the dilution of the label by the underlying LPS makes it difficult to attain high specific radioactivity. This can be avoided in short-term labeling experiments. The filter is removed from its pad, 2. 1 ml of labeling solution in a Petri dish. ) After 30 minutes, the excess fluid can be removed with a pipet.
Add 50 pI to 1 ml of cells suspended at lOXiml to give a final concentration of 500 pgiml NTC. Incubate at 22°C for 20 minutes with shaking. Step 3: Stop mutagenesis. The cells are centrifuged at 300 g for 3 minutes and the pellet resuspended in 10 ml HLS medium. The guanine in the medium inactivates any remaining NTG. The suspension contains lo7 visible cellsiml. 1% of the cells are viable after mutagenesis due to the presence of lethal mutations in most of the cells. Viability varies between and lo-' under these conditions, so the 10-ml suspension contains 103-104 viable cellsiml.
1951). J. Exp. Zool. 118, 407-418. Sussman, M. (1954). J. Gen. Microbiol. 10, 110-120. 2. CULTIVATION OF Dictyostelium Sussman, M. (1961). J . Gen. Microbiol. 25, 375-378. Sussman, M. (1963). Science 139, 338. Sussman, M. (1966). Methods Cell Physiol. 2, 397-410. , and Lovgren, N . (1965). Exp. Cell Res. 38, 97-105. , and Kim, H . (1978). Exp. Cell Res. 116, 217-228. Sussman, R. , and Sussman, M. (1963). J . Gen. Microbiol. 30, 349-355. Sussman, R. , and Sussman, M. (1967). Biochem. Biophys. Res.
Dictyostelium discoideum: Molecular Approaches to Cell Biology by James A. Spudich (Eds.)