1.20.2010 lecture

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1.20.2010 lecture

  1. 1. 1.20.10<br />cysteine – creates disulfide bonds. They occur when the proteins are processed in the golgi apparatus and are either secreted or transported to the plasma membrane. <br />Consider it hydrophilic than hydrophobic. <br />C and H bonds make amino acids hydrophobic.<br />Cell culture:<br />Epithelial – skin, intestines – cells held together and held on exc matric very tightly. Hard to break apart, but can be done. <br />Mesenchyal – blood/bone/smooth muscle. An exc matrix that is widespread and is connective is a mesenchyal. <br />Primary tissue has yielded cell lines – characteristics of the primary tissue but are continually dividing without reaching senescence. <br /><ul><li>controlled environments
  2. 2. reproduce quickly
  3. 3. reproducible – experiments are readily reproduced by the investigator or by someone else who wants to verify the results.
  4. 4. Cheaper and easier than using animals. Cost savings.
  5. 5. Reductionist approach – isolate cells from animal and we can reduce the environment to very simple questions. Allows us to look at very specific questions but can be a disadvantage, taking cell out of normal environment so there is possibility that the cells wont behave the same as in its natural environments
  6. 6. Contamination
  7. 7. Not the cell’s natural environment</li></ul>How to dissociate tissue?<br />-what tissue type? What question to ask?<br /><ul><li>Isolate tissue and consider source – what organ are you taking it from and what animal? Normal tissue? Cancerous? Adult? Younger?
  8. 8. Dissociate tissue – taking tissue and
  9. 9. Chemical – chemical enzymes that break up tissue gently. Trypsin/protinase K/collagenase. These cleave the tight adhesions between cells as well as the proteins that make adhesions with the exc matrix. Break up without damaging cell. Chemical treatments reduces the amont of mechanical dissociation needed. Ca2+ and Mg2+ ions are required for the proteins that hold the proteins that are held to the exc matrix to function. You can remove all the ions from the tissue and that will cause the proteins holding the cells to be released and the cells will release. This Is non-enzymatic.
  10. 10. Mechanical – put in buffer and take a homgenizer and break up tissue into small components.
  11. 11. Combination – often done
  12. 12. Centrifuge – get rid of suspended cell pieces. Membrane bits etc. and take centrifuge material and plate on a primary culture. Will divide until reach confluency – cells have touched one another in a single cell layer. At this stage, cells stop growing b/c receptors required for cells to divide are bound to another saying theyre interacting with the cells and not enough growth factor to continue growing. This can also lead to cell death. But some will live and a secondary culture is created.
  13. 13. Secondary culture – used for replicative experiments and cell lines.
  14. 14. No guarantee they’re all the same cells.
  15. 15. Fluorescene activated cell sorting (facs) allows to mark cells in population with a dye and separate out the cell population.
  16. 16. Run through facs machine. Droplets pass through and a laser hits the cells. The analyzer determines whether the marker Is present or not. if labeled, analyzer imparts a negative charge on cell and moves through the charged plates (+ or -) and deflects into two separate containers. Same for labeled.</li></ul>Flow cytometer – used to sort w/out fluorescent dyes.<br />Blood cells are not adherins but suspended cells.<br />Oncongenic transoformation is characterized by the growth of cells on top of one another. Cancerous mutations. Now the cells have continued dividing and form colonies. Not as dependent on substrate. Not dependent on being adherent to the substrate. <br />Out of the confluent culture, others have died and others have transformed. Used to make cell lines. <br />Stem cells are cells that can be isolated from primary tissue. These are not oncogenic or transformed. These cells are multipotent – cells of a tissue type that continue to divide throughout life giving rise to all cells making up that tissue.<br />In deep layers of dermis, there are epithelial cells and some are stem cells. Each generation is a new stem cell and a new cell of that tissue. Pluripotent stem cells – specifically found in endercell mass of embryo which gives rise to all cell types of all tissues.<br />The media cells grow in is important. <br />Need all essential amino acids/building blocks (vitamins)/salts/miscellaneous/antibiotics used to reduce contamination/dyes/bovine serum – isolated from blood from calves or horse and blood is taken from animal and centrifuged and the organelles are separated and the resulting serum (w/out organelles) is sold.<br />Some organisms yield secondary cultures well.<br />3 stages of growth<br /><ul><li>exponential growth from primary to confluency
  17. 17. cell strain – cells sit for a bit
  18. 18. senescence and many die. (human cells die almost always.) mouse cells enter crisis, and some continue to grow (secondary culture) – immortal, cell lines.
  19. 19. There are human cell lines, but sometimes we must infect with a virus to generate a population of transformed cells. Or take directly from human tumor.
  20. 20. Embryonic stem cells can be used to ask specific questions and without destroying the embryo.
  21. 21. Techniques used to analyze cells in culture.
  22. 22. Prepare sample for imaging. If you have thick samples, you must reduce thickness of sample. A common way is to section it on a microtome. Take fine blade and slice in order to visualize.
  23. 23. Can slice live tissue – organotypic cultures.
  24. 24. Can slice and put in a culture.
  25. 25. Are living and in an intact environment.
  26. 26. 3 things about microscopes you need to know about microscopes.
  27. 27. Resolution -
  28. 28. Magnification – lets you see how large you can view an object while keeping the components as distinct objects. How big can the object be while maintaining objects as distinct?
  29. 29. Contrast – formed by property of cells themselves. Light passes through cells. The cells and organelles are transparent. No definition with organelles and outside environment. Light microscopes. Use dyes to stain to provide contrast and most dyes require you fix and kill cells. To be living you need to manipulate the technology. Add phase contrast or use DIC – interference contrast technology.

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