Manipulation of cell death 11 02-15

Manipulation of Cell Death

1. Pharmacological manipulation of cell death, monitoring response to therapy Mphil 11-2-2015

2. 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

3. How To test Drugs?

4. 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

5. 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

6. Technique and instrument Laminar flow

7. Carbon dioxide incubator

8. Microscope

9. Tissue culture Ware

10. Culture Media Sterilization

11. Cell counting

12. Changing Medium

13. 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

14. 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.

15. 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

16. Monitoring the responses Cell viability Cell proliferation Apoptosis Cell adhesion Chemotaxis Multidrug resistance Endocytosis Secretion Signal transduction

17. Assays to determine viability Dye exclusion- measures membrane integrity and effect of the drug on cell growth Tryptan blue

18. Assays to measure proliferation XTT/PMS Assay This procedure exploits the fact that the internal environment of proliferating cells is more reduced than one of nonviable cells.

19. Specific Stains

20. Fluorescent Stains