Cell Bio Ch 8

Florescence activated cell sorter Uses an antibody coupled to fluorescent dye, the antibody binds to only 1 cell type. Single fluorescent cell gets a negative charge, and the non fluorescent get a positive charge. Accurate to 1 in a thousand cells.

Extracellular membrane is disrupted by proteolytic enzymes. EDTA chelates calcium on which the cell-cell adhesions depend.

Selected cells are dissected from thin tissue slices that are prepared for microscopic examination Cut with laser

Not a convenient source of material. Difficult to purify particular molecules from an intact tissue (too complex). No way possible to do genetic manipulations on the livestock.

Continue to differentiate similar to their origin cells

It provides a more homogeneous population of cells to extract material. Convenient to work with in the lab. Under proper conditions, most plants and animal cells can live, multiply and even differentiate in a tissue-culture dish. In the lab they can be monitored continuously and effect of different media or hormones can be studied. In addition, interactions between two cell types can also be studied. Experiments done on cultured cells are called “in vitro” (“in glass”) in contrast to those done using intact organism “in vivo” (“ in the living organism”).

Culture prepared directly from tissue of the organism.

Primary culture has been repeatedly cultured

Eukaryotic

Cells die after finite number of cell divisions

Cell death after a finite number of cell divisions.

Telomere shortening - cells have turned off production of Telomerase enzyme. As a result telomere shortening occurs with each cell division. Activation of cell –cycle checkpoint mechanism that arrests cell cycle and is called “culture shock”.

Provide telomerase to the cells. Add oncogene to inactivate the checkpoint. Most human cells used in labs are cancer cells

Human cells used in lab, derived from cancer cells

Do not turn off telomerase production. Can grow indefinitely if culture shock is avoided.

Inner mass of early embryo

Can provide specialized cell therapy: replace degenerate muscles in muscular dystrophy. replace insulin secreting cells (Diabetes type I). replace dead nerves in Parkinson’s disease. rejuvenate cardiac muscles in weak heart cells.

If the transplanted cells are genetically different from the patient’s cells it could be rejected by the patient’s immune system. This could be avoided by using cells derived from the patient. If the ES cells are themselves transplanted into a person they can give rise to tumors called teratomas. a

Reproductive cloning-creating another organism or tissue through cloning Or therapeutic cloning- creating generalized personalized embryonic stem cells

Identical to donor cells Can be used for tissue repair Can be used to study inherited disease

Used to separate proteins Mixture of protein passed through a solid porous matrix Different proteins are retarded at different rates by their interaction with the matrix High degree of purity can be obtained

Ion exchange chromatography- proteins separated on basis of surface charge using positive or negative charged beads Hydrophobic chromatography- binds proteins with exposed hydrophobic regions Gel-filtration chromatography- separates proteins by their size Affinity chromatography- proteins separated by their binding properties

To see the localization or purification of proteins Small tag (epitope) targets protein using an antibody Attaches to N-terminus or C-terminus

Flag, GST, 6xHis, c-myc