Flow Cytometry -Introduction and applications

Question 1

What is flow cytometry?

Answer 1

A technique which allows us to simultaneously measure several physical characteristics to a single cell in suspension.

Done by light scatter and fluorescence

Question 2

What is the definition of flow cytometry?

Answer 2

Measuring properties of cells in flow

Question 3

What is the definition of flow sorting ?

Answer 3

Sorting (separating ) cells based on properties measured in flow.

This is also called Fluorescence-Activated Cell Sorting (FACS)

Question 4

What information can flow cytometer tell us about a cell?

Answer 4

• Relative size
• Relative Granularity/Internal Complexity
• Relative fluorescence intensity (using markers)

Question 5

What markers can be measured ?

Answer 5

• Cell surface receptors
• Intercellular cytokines
• DNA-Apoptosis/cell cycle/viability

Question 6

What are two methods of visualisation?

Answer 6

• Flow cytometry

- Fluorescence Microscopy

Question 7

What technique can we use to quantitate cells?

Answer 7

-We can use flow cytometry which can also quantitate the fluorescence

Question 8

How does a flow cytometry work?

Answer 8

Fluidics :
-Cell in suspension flow in a single file through.

Optics:
-An illuminated volume where they scatter light ,emit fluorescence that is collected and filtered.

Electronics:
This is converted into digital values that are stored on a computer
(Then analysed )

Question 9

Outline the different processes involved in flow cytometry

Answer 9

Light source 
Flow chamber 
Optical system
Light detectors
Computer

Question 10

Describe the Fluidics phase

Answer 10

• The cells need to be in suspension flow in single file.
• The sample is injected into sheath fluid as it passes through small orifice (opening)
• Sample fluid flows in a central core that does not mix with the sheath fluid (Laminar flow)

-Introduction of a large volume into a small volume
(Hydrodynamic Focusing)

Question 11

Describe the Light sources for the Optic stage.

Answer 11

Light sources are lasers:

• Single wave length of light (a laser line ) or mix of wavelengths
• Can provide light (milliwatts -watts)
• Inexpensive air-cooled units or expensive water cooled units
• emit coherent light (single frequency)

Question 12

Describe light scatter

Answer 12

This happens when light hits the cell.
-Most common wavelength of laser is 488nm.

• The light becomes scattered in a forward direction (FSC) and this is proportional to cell size
• 90 degrees light scatter proportional to granularity (SSC) Side scatter

Question 13

Describe dot plot

Answer 13

X axis is forward scatter (size)
Y axis side scatter (granularity )

Each dot is a cell.
We can identify different type of cells based on their size and granularity.
The scatter shows this information

Question 14

What is Granularity ?

Answer 14

This is a cells internal complexity

Question 15

Describe the passage of Laser-based flow Cytometry

Answer 15

1.Flow cell - cells leaving the nozzle tip are hit by the laser

2.The cells have been labelled using fluorochromes
They emit light in different colours .
3.There is an overlap in emission spectrum of each fluorochrome.

3.Complex system of mirrors and filters is used to restrict the amount of light hitting the PMT (photomultiplier tube ) = we can differentiate the fluorescence from each fluorochrome separately

Question 16

What is the electronic stage of flow-cytometry?

Answer 16

• This is the final stage
• The PMT will convert light signals into digital signals.
• Analogue- digital conversion

Question 17

What are the key differences between flow cytometry and fluorescence microscopy?

Answer 17

• Fluorescence microscopy (FM) has fields which means its not quantitative as you would need to look at many fields to quantitate cells accurately.
• Flow cytometer(FC) can analyse 1000s every second

Rare cells can be quantitated accurately in FC

Brightness of cells is difficult by eye using FM but the machine in FC will do it and quantitate the amount of fluorescence each cell

Question 18

How does fluorescence happen?

What is this process called?

Answer 18

A laser hits a fluorochrome ,exciting it at a wavelength .It then goes back to to its unexcited state and emits fluorescence at a higher wavelength.
This is called excitation and emission spectrum

Question 19

What is stokes shift ?

Answer 19

The energy difference between the lowest energy peak of absorbance and the highest energy of emission

Question 20

What are some different fluorochromes and dyes ?

Answer 20

FITC (Fluorescein isothiocyanate)- Green
PE (Phycoerythrin)-Orange
PerCP (Peridinin Chlorophyll Protein)-Red

Question 21

What are the wavelengths which each of these fluorochromes emit /excited ?

Answer 21

FITC - Excited at 488 and emits 520
PE-Excited at 488 and emits 580
PerCP- Excited at 488 and emits at 620

Question 22

What is fluorescence ?

Answer 22

Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation

Question 23

Why can different fluorochromes be used together and what is a benefit of this ?

Answer 23

We can use three all excited by the same laser but they emit at different wavelengths.
The emission always overlaps therefore the filters and mirrors will filter out the overlapping light so we can analyse data from all fluorochromes together

Question 24

What are some cells which are in single cell suspension ?

Answer 24

Peripheral blood
Bone Marrow 
Fine Needle aspirate
CSF and other fluids 
Fresh tissue

Question 25

What are the two ways to label cells with monoclonal antibodies /fluorocromes?

Answer 25

Direct :Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes. Indirect: Unconjugated MoABs

Question 26

How is the data displayed when converted from light to digital ?

Answer 26

1.Histogram -One dimensional display (cell number=y axis /fluorescence intensity =x axis (one parameter) 2.Dot plot -Two dimensional side scatter =x axis forward scatter=y axis (Two parameters)- We could potentially identify four populations

Question 27

What are two things could do to the data we have collected ?

Answer 27

Gating | Analysis

Question 28

What is Gating ?

Answer 28

-Two dimensional dot plot Draw a gate around the population we want to look at further. Then we will only display the gated cells

Question 29

What is Analysis?

Answer 29

- Analysis - We will only measure cells satisfying the gate -only analysing one parameter. Using a marker we can see what proportion of the cells that are negative/positive for it .

Question 30

Outline how increasing fluorochromes can increase identified populations

Answer 30

1 fluorochrome =1 population 2 fluorochrome= 4 populations 3 fluorochrome =-8 populations Cells which are positive for all or negative for all

Question 31

How can we analyse cell cycles?

Answer 31

Cellular DNA is detected using a fluorescent dye which binds to DNA Propidium iodide is used -increases fluorescence when binds to DNA Requires permeabilization of the plasma membrane so the PI can enter

Question 32

How is propidium iodide used ?

Answer 32

It is excited by a 488 laser and emits at 620nm

Question 33

What are the different stages of the cell cycle?

Answer 33

G0, G1,S,G2,M

Question 34

What is propidium iodide fluorescence proportional to ?

Answer 34

The amount of DNA in a cell | The G2M phase will have the highest fluorescence and will have double the DNA

Question 35

How can we measure cell viability ?

Answer 35

- PI can not cross a cell membrane - If the PI penetrates the cell membrane the cell is assumed damaged - Cells that are brightly fluorescent with PI are damaged or dead cells

Question 36

Describe apoptosis briefly

Answer 36

Apoptosis is a programmed cell death where the cell goes through regulated process of dying Characteristics are condensation of the chromatin material Blebbing of nuclear material Accompanied by internucleosomal degradation of DNA which gives rise to ladder pattern on DNA gel electrophoresis

Question 37

How can we detect apoptosis ? | What are the three methods ?

Answer 37

By staining using the dye PI -cells are fixed Phosphatidyl serine (this is usually inside but during apoptosis will be on the outside ) can be detected by incubating the cells with fluorescien-labeled Annexin V,PI By staining with 7-aminoactinomycin(cell not fixed)

Question 38

Describe the PI dye method of detecting apoptosis

Answer 38

The Sub -G0 peak This is a peak which is found in apoptotic cells by staining using PI. Issue is not all cells will have this peak and It might all be DNA fragments

Question 39

Describe the Annexin V + PI method of detecting apoptosis

Answer 39

The Phosphatidyl serine normally sits inside the cell. During early apoptosis : -The PS will be outside meaning the Annexin V can bind to it however the cell membrane is in tact The cell is positive for Annexin V but negative for PI During late apoptosis : The cell will be positive for both Annexin V and PI as cell membrane is no longer in tact

Question 40

Describe the 7-Aminoactinomycin D method -7-AAD (apoptosis )

Answer 40

- Excited at 488nm - Emitted at 660nm DNA specific Intercalates in G-C regions Long emission wavelength A single dye -allows us to use two other fluorochromes to evaluate cell surface antigens as well as apoptosis

Question 41

List some applications of flow cytometry.

Answer 41

- Immunophenotyping of leukaemias & lymphomas - Detection of MRD - Stem cell enumeration CD4/CD8 in HIV - Measurement of intracellular cytokines - Study of cell cycle, viability & apoptosis - Measurement of cell proliferation - Assessment of transfection efficiency

Question 42

Describe the fine-tuning of cell sorting

Answer 42

The basics of flow cytometry are present: the cells are in a suspension in a single file, a laser is put on them, causing them to emit light that is a picked up and tells a computer connected to it what type of cell it is. In cell sorting, we can draw a range around the types of cells we want, so the computer will 'sort them out'. The nozzle tip is always vibrating, so much that the stream breaks off into droplets at some point. Within milliseconds of detection of a certain cell, if it is the type that we want, the computer will charge that cell when it is at the end of a droplet, breaking it off. The cell is then collected into a tube by being pulled towards a deflection plate (due to its charge). The rest of the cells the go to be collected for waste. As a result, we get a sample of very purified cells, with endless possibilities in research.

Question 43

Describe the simplest univariate cell cycle method.

Answer 43

In the simplest method, cellular DNA is detected using a fluorescent dye that binds preferentially to DNA. Propidium iodide is most commonly used. It undergoes a dramatic increase in fluorescence upon binding DNA. It requires permeabilization of the plasma membrane.