Lecture 3 - Flow Cytometry II

Question 1

A way to stain for intracellular molecules

Answer 1

Cytokine staining

Question 2

What does cytokine staining do?

Answer 2

Stain for intracellular molecules (EG: of lymphocytes)

Question 3

What needs to be added to a culture of T cells that are to be cytokine stained?

Answer 3

A source of antigen, and of antigen-presenting cells

Question 4

Why does a T cell culture need an antigen and APC source for cytokine staining?

Answer 4

To activate the T cell, so that it produces cytokines

Question 5

Samples often used to provide APCs for T cell cytokine stain

Answer 5

Suspension of splenic cells

B cells infected with a virus

Question 6

How are cytokines visualised in a cytokine stain?

Answer 6

Fluorescent-tagged mAbs

Question 7

Issue with staining with mAbs for intracellular cytokines

Answer 7

Cytokines are within cell. mAbs are outside cell. Need to add agents that

Question 8

Different places that can be cytokine stained

Answer 8

Secreted cytokines - just add fluorescent-mAbs.

Intracellular cytokines - Add an agent that inhibits protein trafficking, fix and permeabilise T cell

Question 9

How can intracellular cytokines be stained for?

Answer 9

Block secretion of cytokines. Fix, permeabilise cell (with mild detergent)

Question 10

What is ‘fixing’ a cell?

Answer 10

Killing a cell, but not lysing it.

Question 11

Isotype controls

Answer 11

Flow cytometry with an antibody which doesn’t bind to target, to measure level of non-specific binding.

Needs to be the same isotype as the staining antibody, and of the same species.

Question 12

Why use isotype controls?

Answer 12

Help to ascertain whether fluorescently-tagged antibody is binding specifically or non-specifically

Question 13

Type of TCR transgenic mouse

Answer 13

gB-specific class I-restricted TCR transgenic (gBT-I mouse)

Question 14

gBT-I mouse

Answer 14

A mouse which only expresses one type of MHC I-restricted T cell receptor, against gB protein of herpes simplex virus.

Question 15

Advantage of gBT-I mouse

Answer 15

TCR is fully-sequenced.

All CD8 T cells have the same TCR

Question 16

CD8 TCR of gBT-I mice

Answer 16

Valpha2, Vbeta8

Question 17

CD45

Answer 17

A protein that can be allelically-expressed as CD45.1 and CD45.2.

Used to identify gBT-I T cells transferred between mice (EG: give CD45.1 T cells to a mouse which expresses CD45.2, identify with an antibody against CD45.1)

Question 18

How do gBT-I T cells appear different from wild-type mouse T cells?

Answer 18

Rearrangement of endogenous genes is inhibited, as a fully-functional TCR is already expressed during development.

Question 19

Pi stain

Answer 19

Propidium iodide stain.
Used to test cell viability.
Is excluded from viable cells.