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