Block A - 3

Question 1

when an activated antigen presenting cell / dendritic cell is introduced what does this allow ?

Answer 1

When an activated antigen presenting cell (APC) / dendritic cell is introduced this allows the B cells and T cells to start interacting. The T cell provides help and allows the B cells to start producing antibodies. This is a difficult thing to do if you are not looking at a live animal.

Question 2

what is in vivo ?

Answer 2

In vivo refers to when research or work is done with or within an entire, living organism. Examples can include studies in animal models or human clinical trials

Question 3

where do immune responses occur ?

Answer 3

in vivo

This occurs in animals and can be visualised, microscopes can be set up for animals when still alive, under anaesthesia. The microscopes are set up in a way so that they do not damage cells, they are often called multiphoton microscopes. They work by targeting the excitation wavelengths from specific layers inside the tissue.

Question 4

what are transgenic animals

Answer 4

Transgenic animals are mouse models that have had their genomes altered for the purpose of studying gene functions and an example is that they contain green fluorescent proteins in some of their tissues on the left.

Question 5

describe in vitro experiments ?

Answer 5

In vitro is used to describe work that’s performed outside of a living organism.

Lots of in vitro assays provide good information in a reductionist, controlled experiment BUT have lost the structure, cellular interactions and complexity of the system as well as requiring manipulation to reveal function (eg. antigen-restimulation). Also has caveats - e.g. complexity of revealing function, cost, technical expertise, ethics.

Question 6

passive immunity ?

Answer 6

Serum from the protected animal could be injected into another animal and then they are challenged by the pathogen, if the animal is healthy this is passive immunity

Question 7

adoptive immunity?

Answer 7

If the animal has spleen cells transferred from the immune animal and remains healthy once the pathogen is introduced, this is adoptive immunity.

Question 8

what does genetic engineering allow ?

Answer 8

us to add and remove specific genes for example missing certain cytokines.

Provides us with lots of models of different diseases ranging from arthritis, asthma to TB, HIV and malaria

Allows us to develop therapeutics by identifying specific molecules important in a given disease so we can target them.

Question 9

why is it important to know stuff about certain cells ?

Answer 9

response to vaccination

effects of drugs

presence of infection

diagnosis of disease

Question 10

important information to know about isolating cells ?

Answer 10

Source of cells? type of cell? Purity necessary?

Influenced about why the experiment is being carried out , for example what cells are required.

Question 11

evaluate number of cells ?

Answer 11

total number? proportion of specific cell-type?

Question 12

cells proliferating ?

Answer 12

Is this antigen-specific? Do they respond to mitogen response?

Question 13

cells viable / dying ?

Answer 13

If dying then why are they dying

Question 14

cells carrying out functions ?

Answer 14

killing? secreting cytokines? Migrating like expected to?

Question 15

how are cells isolated ?

Answer 15

Cells for assay come from a huge variety of sources

Blood can be drawn from humans and many animals using a syringe

However, if lymph nodes are being studied then this would require a dissection.

Question 16

seperation of white blood cells from peripheral blood ?

Answer 16

To separate white blood cells (leukocytes/immune cells) from peripheral blood, use density centrifugation eg. Ficoll-Histopaque. Different densities of samples allow seperation , used to separate peripheral blood mononuclear

Question 17

how can antibodies be used and magnets ?

Answer 17

Using antibodies and magnets, we can be more specific and isolate a certain type or subset of cells for further analysis. Starts of from a heterogenous population and can allow the beads with antibodies to remove heterogenous populations. This is termed negative selection.

Question 18

heamocytometer ?

Answer 18

as a quantification of cells Once we’ve isolated cells from blood, biopsy, lymph node, spleen, etc, we might want to know how many cells we have and what type of cells we’ve purified

Count the number of cells using a hemocytometer:

Question 19

once the number of cells has been calculated how can you tell what are B and T cells ?

Answer 19

To identify how many of those cells are T cells and how many are B cells, often use flow cytometry normally as a percentage. Or can even identify antigen-specific T cells using peptide/MHC. These are tetramers from a recombinant MHC molecule, these bind to T cells which are expressing the correct receptors to the peptide on the MHC molecule.

Question 20

what causes T cells to divide ?

Answer 20

Antigen-specific responses counting on interaction between MHC and co stimulatory molecule and T cell receptor

Mitogen-induced responses
- mitogens activate cells in non-specific manner, driving proliferation

Question 21

what are the 3 ways to measure proliferation ?

Answer 21

Different ways to measure proliferation based on 3 approaches:

Enzyme

Radiation

Fluorescence

Question 22

MTT assay - enzyme ?

Answer 22

Relies on enzymes in the mitochondria ( mitochondrial reducatse) converting salt into coloured product. Not a direct measure of proliferation.Only living cells have active mitochondria. Amount of colour is proportional to number of cells…… which is proportional to amount of cell proliferation.

Question 23

radioactive approach - H thymidine incorporation ?

Answer 23

Add a source of radioactive Thymidine to cell culture

As cells proliferate, they incorporate this 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 24

fluorescent approach ?

Answer 24

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. Typically used in conjugation with a flow cytometry.

Question 25

cell death and dye ?

Answer 25

When cells die, they lose their membrane integrity, this allows vital dyes to enter the cell and stain them.

Eg. Trypan Blue only enters dead cells - can see down conventional light microscope.

Propidium Iodide - becomes fluorescent when binds DNA and this recognises dead cell.

Question 26

chromium ?

Answer 26

The target cell is labelled using chromium, cell killing occurs via CD8.and radioactive chromium is released , which is measured and can measure the cell killing.