Experiment 2 Flashcards
(14 cards)
Necrosis
Cell is damaged
Cell swells then bursts which releases intracellular contents
Release of cell contents can cause damage to other cells
Can activate the inflammatory response
Apoptosis
Mechanism to allow (or tell) cells to die without releasing the cell contents
No intracellular contents are released
Why does apoptosis occur?
Development
Remove old cells (epithelium)
Remove excess cells (end of an immune response)
Kill infected cells (immune response)
Cell stress (lack of growth factors)
Explain morphological changes of apoptosis
Chromatin condenses
DNA fragments
(200bp fragments)
Cytoskeleton and nuclear envelope disassemble
Cell blebs and fragments
Forms apoptotic bodies
Packed cell parts ready for phagocytosis
What is cell blabbing and when does it occur?
Apoptotic bodies that are packaged and ready for phagocytosis. Morphological change during apoptosis
How do we quantify apoptotic cells?
Look at DNA for 200 base pair fragments
Look for activation of apoptosis-specific proteases (caspases)
Electron microscopy for cell blebbing
Stain cells for condensed DNA
What does Syto-24 dye stain?
DNA
What does serum starvation do to cells?
Produces stress on the cells Cell begins to undergo apoptosis within 2-3 days
When analyzing which cell line was affected more by serum deprivation, how do you determine this?
Look at the mean of both + and - serum, and divide each. Look at the fold difference. Whichever has a larger fold increase in apoptosis is the cell line that is more affected.
% apoptotic cells =
total apoptotic cells in X fields/ apoptotic +normal cells * 100
What technique do you use for apoptosis?
Syto24 and counting apoptotic nuclei using a fluorsecent microscope
If we have a sample containing 40 uL of sample, 70 uL of dilutent, and 10 uL of trypan blue… what is the dilution? What about the dilution factor?
Dilution: 1/3
Dilution factor: 3
Suggest two reasons why one cell line may or may not be more affected by serum deprivation than the other cell line.
The 3T3 cells may rely more heavily on the serum for proliferation while HT29 are cancerous cells and may not rely on it as much
HT29 cells may be resistant to apoptosis because they are cancerous and have mutations in apoptotic regulation. This can make them more resistant to cell death.
Define and give the role in apoptosis of the Bak and Bax proteins
Bak and Bax are pro-apoptotic regulators, which means that they induce apoptosis. Bak and Bax are binded to Bcl-2 or BclXl, which prevents them to oligomerize. If a BH3 only protein binds to Bcl2, Bak or Bax is dissociated from Bel-2. Bak or Bax form oligmeric pores which allows from cytochrome-c to pass through the cytosol to activate apoptosis.
