1. FIGURE 13.1 Clonal Cell Yield. Relationship of the cell yield in a clone to the number of population doublings; for example, 20 doublings are required to produce 10 6 cells.

2. FIGURE 13.2 Dilution Cloning. Cells from a trypsinized monolayer culture (a) are counted and diluted sufficiently to generate isolated colonies. When clones form (b; usually larger for optimal isolation), they may be isolated (see also Figs. 13.8, 13.9). If isolation is not required and the cloning is being performed for quantitative assay (see Protocols 20.10, 21.3), then the colonies are fixed, stained (c), and counted. (See also Plate 6a, e.)

3. FIGURE 13.3 Cloning in Microtitration Plates. Cells are seeded at a low enough concentration to give a probability of <1 cell/well, so that some wells will have 1 cell/well, which can be checked by visual examination on the microscope a few hours after plating. Those wells are marked and followed, and when a fall in Ph indicates growth, this is confirmed by microscopic observation and the colony is isolated by trypsinization.

4. FIGURE 13.4 Effect of Glucocorticoids on Cloning. Hydrocortisone analogs enhance the plating efficiency of several cell types. (a) Betamethasone and a human glioma cell line; (b) dexamethasone with SKMES-1 (squares) and A549 (circles) human non–small-cell lung carcinoma cells.

5. FIGURE 13.5 Feeder Layers. Cells are irradiated and trypsinized (or may be trypsinized first and then irradiated in suspension, or treated with mitomycin C) and seeded at a low density to enhance cloning efficiency. (Photo courtesy of M. G. Freshney.)

6. FIGURE 13.6 Cloning in Suspension in Agar. Cultured cells or primary suspensions from bone marrow or tumors, suspended in agar or low-melting-temperature agarose, which is then allowed to gel, form colonies in suspension. Use of an underlay prevents attachment to the base of the dish. (1) Preparation of agar underlay: agar, 1.2%, at 55 ◦ C is mixed with 2× medium at 37 ◦ C and dispensed immediately into dishes, where it is allowed to gel at room temperature or 4 ◦ C. (2) Preparation of agar medium: agar, 1.2%, and UPW are maintained at 55 ◦ C and mixed with 2× medium to give 0.3% agar for cloning. The use of low-melting-point agarose allows all solutions to be maintained at 37 ◦ C, but this agarose can be more difficult to gel. (3) Cells grown in suspension, derived from bone marrow, or trypsinized from an attached monolayer are counted and diluted serially, and the final product is diluted with agar or agarose, and seeded onto an agar underlay.

7. FIGURE 13.7 Cloning in Suspension in Methocel. A series of cell dilutions is prepared as for agar cloning, diluted 1:100 in Methocel medium, and plated into non-tissue-culture-grade dishes or dishes with an agar underlay.

8. FIGURE 13.8 Cloning Rings. Stainless steel rings, cut from tube, in 9-cm glass Petri dish. Porcelain (Fisher), thick-walled glass (Scientific Laboratory Supplies), or plastic (e.g., cut from nylon, silicone, or Teflon thick-walled tubing) can also be used. Whatever the material, the base must be smooth in order to seal with silicone grease onto the base of the Petri dish, and the internal diameter must be just wide enough to enclose one whole clone without the external diameter overlapping adjacent clones.

9. FIGURE 13.9 Isolation of Monolayer Clones. The mature colonies are examined on the microscope, and suitable colonies are selected and marked. The medium is removed, and cloning rings, dipped in silicone grease, are placed around each colony, which is then trypsinized from within the ring.

10. FIGURE 13.10 Isolation of Suspension Clones. Mark the colony as for monolayer, then draw the colony into a Pasteur pipette (or pipettor tip). Transfer the colony to a culture flask, disperse it in medium, and incubate it. Make up medium when cells start to grow.

11. FIGURE 13.11 Suspension Clones of Melanoma, Fibroblasts, and Glia. Cells were plated out at 5 x 10 5 per 35-mm dish (2.5 x 10 5 cells/mL) in 1.5% Methocel over a 1.25% agar underlay. Colonies were photographed after 3 weeks. (a) Melanoma; (b) human normal embryonic skin fibroblasts; (c) human normal adult glia.

12. FIGURE 13.12 Overgrowth in Mixed Culture. Overgrowth of a slowgrowing cell line by a rapidly growing contaminant. This figure portrays a hypothetical example, but it demonstrates that a 10% contamination with a cell population that doubles every 24 h will reach equal proportions with a cell population that doubles every 36 h after only 10 days of growth.