Flow Cytometry

Question 1

What is fluorescence?

Answer 1

Where light is shined onto an object which absorbs the light and then then emits light of a different wavelength.

Question 2

what is a flow cytometer?

Answer 2

A machine that analyses particles (normally cells( in suspension.

Question 3

What can a flow cytometer do?

Answer 3

Count particles.
Tell us how big/granular they are
detect Fluorescent markers on the particles

Question 4

What will a flow cytometer tell us?

Answer 4

What cells we have - how many and of what markers they have.
What the cells are up to
quantification of rare populations
Track changes over time
Break down heterogenous populations of cells into individual populations.

Question 5

What must be required for flow cytometry to work?

Answer 5

Cells are in suspension

The cell/target molecule is fluorescent

Question 6

What cant flow cytometry tell us?

Answer 6

Where the cell is
what other cells/features do the sit next to in the tissue
Information about 1 cell (instead it is about populations)

Question 7

What type of cells are studied using flow cytometry?

Answer 7

Cells - bacteria, eukaryotic cells
Organelles
Chromosomes
Viruses
Organisms - Drosophila embryos
(all cells/particles must be in suspension)

Question 8

What are the key features of flow cytometers?

Answer 8

Laser (light source)
Cells in single file
Light detectors 
mirrors separating wavelengths of light
Computers for data

Question 9

How do flow cytometers detect size?

Answer 9

Forward scatter
Light bends (refracts) when moving through different densities
Therefore when it enters the cell it refracts and then light beam becomes wider
Larger cell = larger width = larger electronic signal

Question 10

How do flow cytometers detect granularity ?

Answer 10

Light passes through cell but some light bounces off granules in all directions which then are detected by side scatter detectors.

Question 11

How can we detect cells apart molecularly?

Answer 11

Identify cells through the molecules present on the outside/inside of the cell.
Antibodies can be created that will stick onto the target molecule (and these antibodies are combined with a fluorescent molecule)
Detect the fluorescence and hence detect the cell.

Question 12

How do we detect different wavelengths of light?

Answer 12

A cell with more than one fluorescent molecule on it will emit more than one wavelength of light
Detectors do not see wavelengths
Wavelengths are separated out using filters called dichoric mirrors

Question 13

How do we detect different colours in flow cytometry?

Answer 13

Light detectors do not see colours.
The more antibodies used
the more fluorochromes we need to detect
the more filters we need to use

Question 14

What does the brighter the cell mean in terms of the electronic signal?

Answer 14

The brighter the cell, the larger the electronic signal

Question 15

What does flow cytometry measure?

Answer 15

Measures changes:
Such as decreasing cell numbers - migration out / cell death
Increasing cell numbers - migration in / proliferation

Question 16

How can cell numbers over time be tracked?

Answer 16

Count the number of cells of each type you have at different times.

Question 17

How can flow cytometry be used to measure proliferation?

Answer 17

Using a dye that can enter cells but doesn’t leave.

Fluorescent molecules enters cells and an ester group is cleaved off.
it cant leave the cell.
the fluorescence is halved each division

Eg. CFSE

Question 18

How can flow cytometry be used to measure cell death?

Answer 18

Use a combination of chemicals:

1. Happy cell
2. Membrane depolarisation (stain with annexin V)
3. Membrane permeability (Stain with 7AAD, propidium iodide, sytox which are DNA binding dyes)

Question 19

What is fluorescence activated cell sorting (FACS)?

Answer 19

When cell sorting you tell the machine what populations of cells you are interested in and then it separates them out for collection.

These cells can then be used for:
culture
sequencing
genetic modification
anything

Question 20

What is the issue when using fluorescence?

Answer 20

It is hard to accurately measure well and can only measure a limited amount of fluorescence.

Question 21

What is high parameter flow cytometry?

Answer 21

A combination of flow cytometry with mass spectrometry which uses elements rather than fluorescent molecules.

Question 22

What are the pros of flow cytometry?

Answer 22

– Lots of data (up to millions of cells per sample)
– Takes hours
– Phenotype and quantify rare populations of cells – Can track changes with time
– Can break down a heterogeneous population of cells into individual populations

Question 23

What are the cons to flow cytometry?

Answer 23

– No anatomical data
– Can be difficult to get cells into a solution
– Can only look at populations, not single cells