Chapter 6

1. Chapter 6 Manipulating Cells in Culture

2. Advantages of working with cultured cells over intact organisms • More homogeneous than cells in tissues • Can control experimental conditions • Can isolate single cells to grow into a colony of genetically homogeneous clone cells

3. Growth of microorganisms in culture • Examples: E. coli and the yeast S. cerevisiae • Have rapid growth rate and simple nutritional requirements • Can be grown on semisolid agar • Mutant strains can be isolated by replica plating Yeast colonies

4. Growth of microorganisms in culture

5. Replica plating

6. Growth of animal cells in culture • Requires rich media including essential amino acids, vitamins, salts, glucose, and serum • Most grow only on special solid surfaces A single mouse cell A colony of human cells Many colonies in a petri dish Figure 6-36

7. Growth of animal cells in culture

8. Primary cells and cell lines • Primary cell cultures are established from animal tissues • Most cells removed from an animal grow and divide for a limited period of time (about 50 doublings), then eventually die • Certain “transformed cells” may arise that are immortal and can be used to form a cell line • Transformed cells may be derived from tumors or may arise spontaneously

9. Establishment of a cell culture Figure 6-37

10. Cell fusion • Two different cells can be induced to fuse thereby creating a hybrid cell (heterokaryon) • Interspecific hybrids may be used for somatic-cell genetics • Certain hybrid cells (hybridomas) are used to produce monoclonal antibodies

11. De Novo and salvage pathways for nucleotide synthesis Figure 6-9

12. Producing a monoclonal antibody to protein X Figure 6-10

13. Chapter 5.5 Purification of cells and their parts

14. Purification of specific cells by flow cytometry Requires fluorescent tag for desired cell Figure 5-34

15. Example: FACS data Figure 5-35

16. Purification of cell parts • Understanding the roles of each each cell component depends on methods to break open (lyse) cells and separate cell components for analysis • Cell lysis is accomplished by various techniques: blender, sonication, tissue homogenizer, hypotonic solution • Separation of cell components generally involves centrifugation

17. Cell fractionation by differential centrifugation Figure 5-36

18. Organelle separation by equilibrium density-gradient centrifugation Figure 5-37