A3

Question 1

Advt/disad to in vitro assays

Answer 1

ad: provide good information in a reductionist, controlled experiment

dis: Lost structure
Cellular interactions
Complexity of the system as Requirs manipulation to reveal function (eg. antigen-restimulation)
Also
Complexity of revealing function, cost, technical expertise, ethics

Question 2

Transfer of
1. Serum
2. Spleen cells
give what type of immunity

Answer 2

1. Passive immunization

2. Adoptive immunization

Question 3

How are white blood cells separated from peripheral blood

Answer 3

Density centrifugation eg. Ficoll-Histopaque
Peripheral blood mononuclear cells on top/
Red blood cells & granulocytes on bottom

Question 4

How can magnets be used to separate antibodies?

Answer 4

1. Heterogenous pop of lymphocytes is mixed w/ antibodies coupled to paramagnetic particles or beads & poured over an iron wool mesh
2. Magnetic field is applied the coupled cells stick to the iron wool -> unlabeled cells are washed out
3. The magnetic field is removed releasing the coupled cells

Question 5

How can MHC:peptide tetramer be used to identify antigen-specific Tc

Answer 5

1. The MHC:peptide tetramer is made from recombinant MHC molecules w/ specific peptides, bound to streptavidin via biotin
2. MHC:peptide tetramers are bound by Tc expressing receptors of the appropriate specificity
3. Flow cytometry can be used to quantify

Question 6

Name 3 ways of measuring cell proliferation

Answer 6

1. Enzymes
2. Radiation
3. Fluorescence

Question 7

The mitogen simulates what type of cells

1. Phytohemagglutinin (PHA)
2. ConA
3. Pokeweed mitogen (PWM)
4. Lipopolysaccharide (LPS)

Answer 7

1. T cells
2. T cells
3. T & B cells
4. B cells (mouse)

Question 8

Describe enzymatic approach to cell proliferation and give example

Answer 8

e.g. MTT assay
Relies on enzymes in the mitochondria converting salt into coloured product.
Only living cells have active mitochondria.
Amount of colour is proportional to number of cells –> which is proportional to amount of cell proliferation

Question 9

Describe Radioactive approach to cell proliferation and give example

Answer 9

eg. 3H-Thymidine incorporation
Add a source of radioactive Thymidine to cell culture
As cells proliferate, they incorporate radioactivity into the DNA of daughter cells, resulting in radioactive cells.
Then measure the amount of cell-associated radioactivity, proportional to the amount of cell division

Question 10

Describe Fluorescent approach to cell proliferation and give example

Answer 10

Can label proteins of cells with fluorescent dye (eg. CFSE = carboxyfluorescein succinimidyl ester)
If cell divides, each daughter cell is half as bright
Allows accurate quantification of the number of cell divisions within the population

Question 11

How can cell killing be measured

Answer 11

radioactivity or fluorescence

• Target cells labelled w/ Na251CrO4 (chromium radionucleotide)
• cytotoxic Tc added to labeled target cells
• killed cells release radioactive chromium