APOPTOSIS AND ANASTASIS, Slides of Cell Biology

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APOPTOSIS – ANASTASIS

22MSBI

REESHMA.K

CELL BIOLOGY

APOPTOSIS  (^) Apoptosis-falling off or falling to death.  (^) It is a natural biological process of programmed cell death in which the cells destroy themselves for maintaining the smooth functioning of the body.  (^) 50 – 80 billion cells die every day in a human adult due to apoptosis.  (^) During this biological process, infected cells, pre-cancerous cells and other cancer cells are eliminated successfully and maintain the balance of cells in the human body.

PREPARATION OF CELLS FOR LIVE CELL IMAGING  (^) Adherent cells- HeLa.  (^) Cleaning and sterilization of glass wares.  (^) Bottom culture dish is coated with Poly-d-lysine , Collagen , Fibronectin before seeding cells.  (^) Cells are seeded and incubated at 37°c – 1 day- to achieve 80% confluency.

LIVE CELL MICROSCOPY  (^) An inverted confocal or epi-fluorescence microscope with environmental control is used.  (^) A 35 mm glass bottom dish of cultured cells are placed on the stage of the microscope.  (^) Cells are maintained at 37 °C and transparent foil is used for preventing evaporation of the medium.  (^) Ph is maintained.  (^) Minimize fluorescence/laser exposure to cells during the imaging process to avoid phototoxicity.

MITOCHONDRIAL FRAGMENTATION,CHROMATIN CONDENSATION AND NUCLEAR FRAGMENTATION  (^) Mitochondria , Nuclei were stained with mitotracker (red fluorescence) and hoechst (blue fluorescence).  (^) Cells are washed ,incubated with culture medium  (^) Fixed with 3.7% paraformaldehyde in phosphate-buffer saline (PBS)  (^) The fixed cells were mounted on glass slide.  (^) Cell imaging – confocal inverted microscope.  (^) Trigger apoptosis , time lapse tracking of cells.  (^) Display of hallmarks of apoptosis-wash and supply the cells with fresh medium.

DETECTION OF MITOCHONDRIAL RELEASE OF CYTOCHROME C  (^) Cells expressing cytochrome c -GFP –stain with Mitotracker-polarized cell mitochondria.  (^) Observe cells using microscope  (^) Trigger apopotosis  (^) cytochrome c -GFP expression is observed , cells are washed  (^) Capture DIC images, alterations in cell Morphology is observed.

CELL SURFACE EXPOSURE OF PHOSPHATIDYLSERINE  (^) Seed cells on glass coverslips to achieve 80% cell confluency  (^) Mitochondria , nuclei were stained with mitotracker (red fluorescence) and hoechst (blue fluorescence).  (^) Trigger apoptosis  (^) Apply fluorescent-labelled - stain annexin v  (^) Washed with 10% fetal bovine serum (fbs)  (^) Fix cells on glass slides and observe them using confocal microscope.

CONCLUSION  (^) We can reverse the apoptotic process at the execution stage “point of no return”, including caspase-3 activation, DNA damage, and cell shrinkage.  (^) Anastasis could also be an important mechanism of tumorigenesis, as some cells display oncogenic transformation after they reversed apoptosis.  (^) The ability of cells to recover from transient induction of cell death may allow tumour cells to escape treatment, and survive and proliferate, resulting in relapse  (^) Enhancing anastasis could be a new therapeutic strategy for inhibiting neurodegeneration and heart failure, while suppressing anastasis as a new way for preventing or treating cancers.  (^) Developing biosensors - track reversal of apoptosis in disease model systems will advance the understanding of the cell survival mechanisms of anastasis, and potential therapeutics to intractable diseases by modulating anastasis.