Download - Manipulation of cell death 11 02-15
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Pharmacological manipulation of cell death, monitoring response to
therapyMphil
11-2-2015
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Etiology of cell death
Major Factors
Accidental Genetic
Necrosis Apoptosis
Necrosis: The sum of the morphologic changes that follow cell death in a living tissue or organ
Apoptosis:a physiological process that includes specific suicide signals leading to cell death
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How To test Drugs?
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Cell culture
Cells, previously growing in a human or animal modified to grow in plastic or glass
Kept in an incubator to stay at body temperature
We use special media with nutrients so the cells can grow and divide
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What can we do with cells?
Test pharmaceutical drugs
Watch disease mechanisms
Design potential treatments
Observe the regenerative process
How do cells and tissues repair themselves after damage from illness or injury?
Observe the developmental process
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Technique and instrumentLaminar flow
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Carbon dioxide incubator
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Microscope
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Tissue culture Ware
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Culture Media Sterilization
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Cell counting
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Changing Medium
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Passaging cells (subculturing cells)
Process of diluting cell number in order to keep cells actively growing
For adherent cells, when they cover the tissue culture dish, they need to be passaged
Otherwise, the cells will become unhealthy and stop growing
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Other miscellaneous Equipment
• Fridge Freezer for storing medium
• Liquid nitrogen Container for cryopreservation of cells
• Incubator for warming up of the medium
• Bench centrifuges to separate out cell pellet.
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Adherent vs Suspension cells for tissue culture
Adherent cells: cells grow in a single layer (called a monolayer) attached to the tissue culture dish Cell growth is limited by available
surface area on which cells can grow
To passage adherent cells, the cells must be released from the dish (done either enzymatically, chemically, or mechanically)
Suspension cells : cells are suspended in liquid as single cells or as free-floating clumps of a few cells To passage suspension cell cultures, a
proportion of the cells in culture are diluted into a larger volume of medium
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Monitoring the responses
• Cell viability• Cell proliferation• Apoptosis• Cell adhesion• Chemotaxis• Multidrug resistance• Endocytosis• Secretion• Signal transduction
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Assays to determine viability
• Dye exclusion- measures membrane integrity and effect of the drug on cell growth
• Tryptan blue
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Assays to measure proliferation
XTT/PMS AssayThis procedure
exploits the fact that the internal
environment of proliferating cells is more reduced than
one of nonviable cells.
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Specific Stains
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Fluorescent Stains