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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 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.