Pharmacological manipulation of cell death, monitoring response to

therapyMphil

11-2-2015

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

How To test Drugs?

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

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

Technique and instrumentLaminar flow

Carbon dioxide incubator

Microscope

Tissue culture Ware

Culture Media Sterilization

Cell counting

Changing Medium

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

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.

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

Monitoring the responses

• Cell viability• Cell proliferation• Apoptosis• Cell adhesion• Chemotaxis• Multidrug resistance• Endocytosis• Secretion• Signal transduction

Assays to determine viability

• Dye exclusion- measures membrane integrity and effect of the drug on cell growth

• Tryptan blue

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.

Specific Stains

Fluorescent Stains