Cytotoxicity Assay

Question 1

What questions do you need to ask when doing a cytotoxicity assay?

Answer 1

What are the endpoints you’re measuring(viability vs proliferation)
Is this a suitable cell model for the question you’re asking(chemotherpy target need proliferating cells)
Is it affecting other types of cell populations(stromal cells)

Question 2

How to measure if cells are alive

Answer 2

Trypan blue dye stains cells of the membrane is permeable(cells are dead)
Or cell metabolic activity measured and cell counted using a haemocytomter
Can measure different cellular marker of viability

Question 3

How to measure if cells are proliferating?

Answer 3

Clonogenic assay
Plate them on methylcellulose medium a defined number of cells are plated before treatment and after a period of time colonies are counted and compared to control.

Question 4

What are the endpoints of cytotoxicity assay? Are they alive or not assay?

Answer 4

1. Use dyes to stain cellular components
2. Detect constitutive enzymes or cellular components
3. Measure metabolic function

Question 5

Give example of using stains to measure the cellular components

Answer 5

Annexin V binds to phosphotidyl serine and prodium iodide stains DNA in apoptotic cells
Trypan blue and counting cells

Question 6

Example of detection of specific constitutive enzymes

Answer 6

Bioluminescent assay. Add luciferin and luciferase and with o2 and ATP IT’ll glow
Can measure:
1.ATP
2. Lactate dehydrogenase
Pentose phosphate pathway

Question 7

Example of measuring metabolic activity?

Answer 7

Using tetrazolium salt(yellow) which will be converted to formazen(red) by dehydrogenase enzyme.
Almar rest-uses nonfluorescent dye(blue) it’s reduced to a fluorescent pink because of mitochondrial activity

Question 8

Measuring the dead cells at a certain time using apoptosis assay. What are the different ways to detect apoptosis?

Answer 8

1. TUNEL assay-measured fragmented DNA by dutp fluorescent or biotinylated molecule(activated by TDT enzyme and add strep avidin to detect) binds to 3’OH of DNA
2. annexinV and propidium iodide labellling.
3. EffectorCaspases detection
4. DNA Detection ladder
5. H2AX phosphorylation
6. Mitochondrial function
7. PARP detection, involved in DNA repair, cleaved when there is apoptosis
8. Electron microscopy

Question 9

How does measuring mitochondrial function in apoptotic cells work? And give an example of a dye used?

Answer 9

When cells apoptose they lose transmembrane potential, so a cationic lipophilic fluorochrome Will bind to the negative charge molecules.
An example of a dye is rhodamine 123, JC1 diffuses into mitochondria and becomes green in apoptotic cells.

Question 10

What are some ways assays are optimal?

Answer 10

Fast assays that detect a signal such as ATP or NADPH are better than the ones that detect metabolic activity because artifacts are reduced. They provide a snapshot of an average response of cells. Good model? Mimicking environment?