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Molecular methods of cell culture III

Molecular methods of cell culture III. Apoptosis  Programmed cell death A physiological mechanism to eliminate excess, damaged or dangerous cells from an organism without damaging surrounding cells and tissues  Necessary for normal embryogenesis

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Molecular methods of cell culture III

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  1. Molecular methods of cell culture III

  2. Apoptosis •  Programmed cell death • A physiological mechanism to • eliminate excess, damaged or • dangerous cells from an organism • without damaging surrounding cells and tissues •  Necessary for normal embryogenesis •  Maintenance of tissue homeostasis

  3. Apoptotic morphology •  Membrane blebbing •  Aggregation of chromatin at the nuclear membrane • Ends with fragmentation of cell into small bodies • Begin with shrinking of cytoplasm and condensation of nucleus • Formation of apoptotic bodies • Mitochondria become leaky due to pore formation • involving proteins of the bcl-2 family

  4. Apoptosis vs Necrosis apoptosis Inflammation

  5. Apoptotic analysis parameter DNA strand break Altered nucleus morphology Reduced DNA content

  6. Detection of apoptosis in cell culture Increased low molecular weight DNA in apoptotic cells

  7. Apoptosis vs Necrosis

  8. Apoptotic morphology

  9. Apoptotic morphology Normal Apoptosis Chromatin Condensation DAPI stain Journal of Gastroenterology and Hepatology 22 :(2007) 738–748 Ibio.com

  10. DNA fragmentation Electroporation or Transfection of apoptotic molucules UV Irradiation nucleus DNA break DNA fragmentation

  11. DNA fragmentation

  12. IAP: apoptosis inhibitory protein

  13. In situ labelling of DNA break Terminal dideoxynucleotidyl transferase

  14. APO- BrdU TUNEL Assay DNA strand break caused by endonucleasea produced by apoptosis process Add BrdUTP’s to 3’-OH DNA strand breaka usingTgT enzyme as catalyst Fluorescented antibody labeling of BrdUTP attached to 3’OH DNA strabd break

  15. Griffin et al.Cancer Cell International 2007 7:10  

  16. Flowcytometry analysis of cell culture Phenotype analysis DNA analysis Apoptotic analysis gene functional study

  17. Flow cytometry

  18. Flow cytometry Emission Emission Excitation DATA analysis

  19. Apoptotic analysis by flow cytometry Clinical and Experimental Immunology,2005, 140: 360–367

  20. cyclinA CDK2 cyclinA cyclinE CDK1 CDK2 cyclinD CDK6 Cell cycle analysis of cell culture S G2 G1 M cyclinD CDK4

  21. apoptosis subG1 subG1

  22. apoptotic cells G1 G2/M S Journal of Gastroenterology and Hepatology 22 :(2007) 738–748

  23. Anti-Fas-induced apoptotic L929 cells - Morphology, Lysotracker Red & SG uptake overlay http://www.youtube.com/watch?v=iPZpubaiZPo&NR=1&feature=endscreen TNF-induced necrotic L929 cells - Morphology, mito. potential & SG uptake overlay http://www.youtube.com/watch?v=JKaEFzsj3l0&feature=relmfu

  24. Migration assay of cell culture  Embryonic development  Cancer invasion and metastasis  Chemo attractant of immune cells  Tissue repair  Angiogenesis www. biochemweb.org

  25. lower chamber upper chamber membrane with different pore size drug treatment or expression of foreign genes lower chamber upper chamber membrane with different pore size

  26. Drug treatment manupalation of foreign genes membrane with different pore size cell migration through membrane fluorescent observation colormetric observation

  27. Control Experimental Experimental treatment

  28. Vaccinia virus induced cell migration http://www.youtube.com/watch?v=NYvgkMUdisU&feature=related

  29. Fluorescent Confocal microscope DNA transfection Immunofluorescent staining Expressionn od foreign gene of interest

  30. a-SMA Actin Merge

  31. The role of MMPs in vascular smooth muscle cell migration.  Phalloidin stain (red) to show actin and Hoechst stain (blue) for nuclear stain.  Johnson C, Fini ME, Galis ZS. 2002. muscle cell (SMC) migration and attachment to extracellular matrix”, FASEB Journal 16(4):A590.  

  32. 3 Dimentional cell culture • Experimental Therapeutics • Metabolism and metabolic environment • Mathematical modeling • Invasion and metastasis • Angiogenesis • Experimental tissue modeling • Embryoid bodies http://www.youtube.com/watch?v=N8Q4zscRWWs Am. J. Physiol. 273 (Cell Physiol. 42): C1109–C1123, 1997

  33. Culture system of monolayer and Air Liquid Interface Air Liquid Interface Monolayer Pulmonary epithelial cells S.G. Klein et al. / Toxicology in Vitro 25 (2011) 1516–1534

  34. Triculture system with endothelial cells to study the inflammatory effect And pulmonary cell communication in vitro S.G. Klein et al. / Toxicology in Vitro 25 (2011) 1516–1534

  35. cell cocultures Plates for coculture of cells. The plates contain a membrane that allows separation of different cell types or media.

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