Lecture 5

Question 1

What is flow cytometry?

Answer 1

The analysis of cellular characteristics as the cells pass singly in a fluid suspension through a beam of light.
Beam of light = laser.

Question 2

What different aspects of the cell can we look at using flow cytometry?

Answer 2

Cell size
Cell shape
Cell components (using fluorescent tags)
Cell function (using fluorescent tags)

Question 3

What is the forward scatter detector measuring?

Answer 3

Usually a measure of size

Question 4

What is the 90° of side scatter detector measuring?

Answer 4

A measure of internal complexity.

Question 5

How can different cell types in mixtures be identified?

Answer 5

By light scatter from the laser.
Different cell types have different types of light scatter.
Dot plot.

Question 6

How is fluorescence detected?

Answer 6

Fluorescent antibodies added to cell surface.
Fluorescent detectors (photomultiplier tubes).

Question 7

Describe flow cytometry optics.

Answer 7

Turns light into an electrical signal.
Dichroic filter mirrors allow for certain wavelengths of light through or reflect them to get a neat and defined wavelength.

Question 8

What are the common applications of flow cytometry?

Answer 8

DNA analysis
Phenotypic analysis
Isolation of small populations (cell sorting)

Question 9

Describe detection of apoptosis.

Answer 9

During apoptosis nucleases degrade DNA so it can elute from the cell.
Treat cell culture with cytotoxic agent that causes apoptosis.
Can see G1/0 peak and there is a sub G1 peak where apoptotic cells are.

Question 10

Describe cell sorting.

Answer 10

Cells travel in a stream through a vibrating nozzle producing a stream of droplets.
Cells are analysed at the laser and a sort decision is made.
When the cell reaches the break off point the stream is charged.
Charged droplets are deflected by electrostatic fields from high voltage plates.
Cells are collected in tubes.