Flow Cytometry: Introduction And Applications

Question 1

What is flow cytometry?

Answer 1

Technique that simultaneously measures several physical characteristic belonging to a SINGLE CELL in SUSPENSION using LIGHT SCATTER AND FLUORESCENCE.

Question 2

What is flow sorting?

Answer 2

FACS (fluorescence activated cell sorting) - separates cells based on properties in flow

Question 3

What properties can be measured by flow cytometry?

Answer 3

Relative size of cell. Relative granularity/complexity. Relative fluorescence (after adding fluorescence e.g. to surface receptors)

Question 4

Why is fluorescence microscopy disadvantageous compared to flow cytometry for visualisation?

Answer 4

Only few cells at a time can be viewed whereas flow cytometry measures 1000’s at once.
Cannot accurately quantify or look for rare cells
Microscopy is subjective when estimating level of fluorescene emitted.

Question 5

What are the 3 stages of flow cytometry?

Answer 5

Fluidics - cells in suspension in single file
Optics - scatter light and emit fluorescence
Electronics - convert to digital values (store and analyse)

Question 6

How are cells forced into single file in the fluidics stage?

Answer 6

Inject sample fluid into central core surrounded by sheath fluid (do not mix) - laminar flow of sample fluid

HYDRODYNAMIC FOCUSING - large volume into small forces into single file

Question 7

What happens in fluidics after cell suspension is in single file?

Answer 7

Single file so they are excited by laser light source one by one. Stained cells will emit fluorescence.

Forward and side scatter is then detected in optics

Question 8

What is important about the wavelength used in the laser to excite the fluorchrome?

Answer 8

Coherent light - light is emitted ALWAYS at single frequency

• Usually single wavelength, more rarely mixture of wavelengths

Question 9

What does forward scatter (FSC) identify?

Answer 9

Size of cells

Question 10

What does side scatter (SSC) at 90 degrees show?

Answer 10

Granularity of cells

Question 11

What do dots represent on population dot plot?

Answer 11

single cell

Question 12

What happens when cells are labelled with more than 1 type of fluorochrome?

Answer 12

All excited at same level but emits lights of different levels which pass through mirrors and filters to avoid overlap before hitting respective PMT (photomultiplying tube) which differentiates fluorescence from each fluorochrome all at once despite some overlap from being excited at the same level.

Question 13

How is the signal from detectors converted in electronics stage?

Answer 13

Analog - digital conversion

- PMT converts light signals to digital signals

Question 14

How does fluorescence from fluorochromes occur in flow cytometry?

Answer 14

Excitation - emission
Laser must hit the fluorochrome and excite it at one wavelength. When returning to original wavelength, fluorescence is emitted at a higher wavelength.

Question 15

What is stokes shift?

Answer 15

Energy difference between lowest energy peak of absorbance and highest energy of emission.

Question 16

Name the fluorchromes for green, orange and red fluorescence?

Answer 16

Green = FITC (fluorescein isothiocyanate)
Orange = PE (phycoerythrin)
Red = PerCP (peridinin chlorophyll protein)

Question 17

Examples of single cells in suspension

Answer 17

peripheral blood
bone marrow
csf + other fluid
fresh tissue (needs to be disaggregated first)

Question 18

How are cells labelled with fluorchromes?

Answer 18

using monoclonal antibodies

Question 19

What is direct and indirect labelling?

Answer 19

Direct - monoclonal antibodies are preconjugated to fluorochromes

indirect - unconjugated monoclonal antibodies (secondary antibody has the fluorochrome)

Question 20

What are the 2 forms of data display? and which one shows more information + why

Answer 20

Histograms - shows one parameter + 2D

Dot plot - 2D but SSC and FSC = more tan one paramter

Question 21

What are the steps in data presentation? + brief explanation of each

Answer 21

Gating and analysis

gating - gate a population of cells e.g. leukocytes. Converter separates into subset populations. Identify cell of interest using fluorochrome.

analysis - histogram of the one population showing that one fluorochrome. Convert histogram to box plot with quadrant to identify proportions within the different populations of target cells e.g. CD3 within CD8 cells

Question 22

What do the peaks on histograms represent in flow cytometry?

Answer 22

+ve and -ve peak. 1st peak is -ve and 2nd peak is +ve since fluorescence is emitted at higher level. How big the peak is shows the number of cells that is +ve/-ve.

Question 23

How can flow cytometry shows be used to investigate cell cycle?

Answer 23

Cellular DNA is detected using fluorescent dye that binds preferentially to DNA.

Question 24

Which fluorescent dye is used in investigating cell cycle stages? + why?

Answer 24

Propidium Iodide - goes through dramatic increase in fluorescence upon binding to DNA

Question 25

What does the excitation emission spectrum show for propidium iodide (PI)?

Answer 25

Excites at blue, Emits in red region

Question 26

How is cell viability checked in cell populations?

Answer 26

Undamaged cells are viable and PI cannot pass the cell membrane. If PI can penetrate, the cell is damaged and will brightly fluoresce.

Question 27

What is apoptosis?

Answer 27

Highly regulated process of programmed cell death

Question 28

What are the genetic material characteristics of apoptosis?

Answer 28

Condensation of chromatin and blebbing of nuclear material

Internucleosomal degradation of DNA (ladder on DNA gel electrophoresis)

Question 29

What are the 3 detection methods for apoptosis?

Answer 29

1) Stain with dye PI - will enter the dying cells
2) Fluorescein labelled Annexin V binds to phosphatidyl serine (normally on inside of membrane - outside in apoptosis) + PI
3) Stain cells with 7-aminoactinomycin

Question 30

What are the uses of PI?

Answer 30

• proportion of cells in diff stages of cells cycle as well as amount of DNA in each stage of cell cycle
• Check for cell viability
• Detect apoptosis

Question 31

How do histograms show apoptotic cells?

Answer 31

PI attaches to DNA in sub G0 cells - there will be a peak before G1 phase called G0 phases if there is apoptosis - not reliable method incase G0 cells aren’t dying

Question 32

Why is annexin - FITC and PI used in combination to identify apoptosis?

Answer 32

Early and late apoptotic/necrotic cell differentiation

• only annexin FITC (green dye) binds to phosphotidyl serine on early apoptotic cell
• PI also enters cells as membrane no longer in tact in late apoptotic cells

Question 33

What are the 3 different populations identified with annexin FITC and PI combination?

Answer 33

double negative = live cells
annexin FITC = early apoptotic
double positive = necrotic

Question 34

What is the benefit of using 7-AAD

Answer 34

Not only FITC and PE labelled but also 2 other fluorchromes used so can evaluate cell surface antigens as well as identifying apoptic cells

Question 35

What are the steps in cell sorting?

Answer 35

Physical separation of defined cells in populations without cultivation

Droplet containing one cells breaks off when stream reaches certain point and charge the stream.

Deflection tubes sort charged droplets into diff fractions