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Molecular methods of cell culture III. Apoptosis  Programmed cell death  A physiological mechanism to eliminate excess, damaged or dangerous cells from.

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Presentation on theme: "Molecular methods of cell culture III. Apoptosis  Programmed cell death  A physiological mechanism to eliminate excess, damaged or dangerous cells from."— Presentation transcript:

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 DNA strand breakAltered nucleus morphology Reduced DNA content Apoptotic analysis parameter

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

7 Apoptosis vs Necrosis

8 Apoptotic morphology

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

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

11 DNA fragmentation

12 IAP: apoptosis inhibitory protein

13 Terminal dideoxynucleotidyl transferase In situ labelling of DNA break

14 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 APO- BrdU TUNEL Assay

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 Excitation Emission Flow cytometry DATA analysis

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

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

21 apoptosis subG1

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

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

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

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

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

27

28 ControlExperimental treatment

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

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

31 Actin  -SMA Merge

32 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.

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

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

35 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

36 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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