Transcript 投影片 1
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 Maintenance of tissue homeostasis 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 Apoptosis vs Necrosis apoptosis Inflammation Apoptotic analysis parameter DNA strand break Altered nucleus morphology Reduced DNA content Detection of apoptosis in cell culture Increased low molecular weight DNA in apoptotic cells Apoptosis vs Necrosis Apoptotic morphology Apoptotic morphology Normal Apoptosis Chromatin Condensation DAPI stain Journal of Gastroenterology and Hepatology 22 :(2007) 738–748 Ibio.com DNA fragmentation UV Irradiation Electroporation or Transfection of apoptotic molucules nucleus DNA break DNA fragmentation DNA fragmentation IAP: apoptosis inhibitory protein In situ labelling of DNA break Terminal dideoxynucleotidyl transferase APO- BrdU TUNEL Assay DNA strand break Add BrdUTP’s to caused by 3’-OH DNA strand breaka endonucleasea usingTgT enzyme produced by apoptosis process as catalyst Fluorescented antibody labeling of BrdUTP attached to 3’OH DNA strabd break Griffin et al. Cancer Cell International 2007 7:10 Flowcytometry analysis of cell culture Phenotype analysis DNA analysis Apoptotic analysis gene functional study Flow cytometry Flow cytometry Emission Emission Excitation DATA analysis Apoptotic analysis by flow cytometry Clinical and Experimental Immunology,2005, 140: 360–367 Cell cycle analysis of cell culture cyclinA CDK2 cyclinE CDK2 cyclinD cyclinA CDK6 CDK1 cyclinD CDK4 apoptosis subG1 subG1 apoptotic cells G1 S G2/M Journal of Gastroenterology and Hepatology 22 :(2007) 738–748 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 Migration assay of cell culture Embryonic development Cancer invasion and metastasis Chemo attractant of immune cells Tissue repair Angiogenesis www. biochemweb.org lower chamber upper chamber membrane with different pore size lower chamber membrane with different pore size drug treatment or expression of foreign genes upper chamber Drug treatment membrane with different pore size colormetric observation manupalation of foreign genes cell migration through membrane fluorescent observation Control Experimental treatment Experimental Vaccinia virus induced cell migration http://www.youtube.com/watch?v=NYvgkMUdisU&feature=related Fluorescent Confocal microscope DNA transfection Immunofluorescent staining Expressionn od foreign gene of interest a-SMA Actin Merge 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. 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 Culture system of monolayer and Air Liquid Interface Monolayer Air Liquid Interface Pulmonary epithelial cells S.G. Klein et al. / Toxicology in Vitro 25 (2011) 1516–1534 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 cell cocultures Plates for coculture of cells. The plates contain a membrane that allows separation of different cell types or media.