Lecture 3 - Flow Cytometry, Transgenic Mice

Question 1

Describe how flow cytometry may be used to detect cytokines

Answer 1

1. Harvest T cells and culture in vitro, expose to APC and antigen to cause it to become activated2. Add an inhibitor that blocks cytokine secretion. Cytokines accumulate in the T cells3. Add agents to fix and permeabilise (w/ mild detergents) the T cells. Abs can not move inside the cellNB Ab conjugated to a fluorochrome4. Perform flow cytometry & formulate a scatter plot

Question 2

Describe the process of creating a transgenic mouse

Answer 2

1. DNA sequence for TCR of desired specificity injected into embryonic stem cells of mice2. DNA incorporates and overrides all other endogenous genes for TCRs in this mouse3. All T cells in this mouse will express the TCR of desired specificity

Question 3

What is the benefit of using transgenic mice in immunology?

Answer 3

T cells may be harvested from the transgenic mouse and injected into WT mouseThis allows the investigation of T cell responses to the desired AgNormally the desired T cells are very rareBy increasing their number, they are easier to study