Flow cytometry

Question 1

What is flow cytometry?

Answer 1

• technique which simultaneously measures several physical characteristics belonging to a single cell suspension
• this is done by light scatter and fluorescence

Question 2

What does a flow cytomer tell us about a cell?

Answer 2

1. its relative size
2. its relative granularity/internal complexity
3. its relative fluorescence intensity

Question 3

What are some things we can measure using flow cytometry?

Answer 3

1. cell surface receptors
2. cell surface enzymes
3. intracellular cytokines
4. apoptosis

Question 4

What is an alternative technique to flow cytometry?

Answer 4

• fluorescence microscopy
• hard to quantitate cells accurately compared to flow cytometry
• hard to quantitate rare cells.
• brightness is different and hard to detect with eye but in flow cytometry the machine does it for you

Question 5

what are 3 components to cytometry?

Answer 5

1. fluidics:
   -cells in suspension
   -flow in single-file through
2. optics:
   - an illuminated volume where they scatter light and emit fluorescence that is collected, filtered
   3.electronics :
   and converted to digital values that are store on a computer.

Question 6

How does the flow cytometry actually work?

Answer 6

1. light source
2. flow chamber
3. optical system
4. light detectors
5. computer

Question 7

What happens in fluidics?

Answer 7

• need to have all cells in suspension flow in single file
• accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 micrometre)
• sample fluid flow 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 8

What happens in optics - light source?

Answer 8

Laser

• single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
• can provide from milliwatts to watts of light
• can be inexpensive, air-cooled units or expensive, water -cooled units
• provide coherent light (single frequency)

Question 9

What is channel layout for laser based flow cytometry?

Answer 9

• overlap in emission spectrum of fluorescence
• filters and mirrors restrict the amount of light hitting photo-multiplier tubes (PMTs) so we can differentiate the fluorescence.
• PMT 1-4 order : blue, green, yellow, red

Question 10

what happens in electronics phase?

Answer 10

• processing of signals from detectors

- PMT convert light signals to digital signal (analogue digital signal)

Question 11

How does fluorescence happen?

Answer 11

-laser hits fluorochrome and its excited at one wavelength and when it goes back to its unexcited state it emits fluorescence at a higher wavelength.

Question 12

Define stroke shift

Answer 12

• LA HE

- energy difference between the lowest energy peak of absorbance and the highest energy of emission.

Question 13

Give examples of fluorochromes/ dyes

Answer 13

1. fluorescein isothiocynate (FITC) = green , excited = 488 and emitted = 520
2. Phycoerythrin (PE) = orange , excited = 488 , emits = 580
3. Peridinin chlorophyll protein (PerCP) = red
   excited = 488 , emitted = 620.
   => all excited by same laser

Question 14

What are methods of labelling?

Answer 14

1. direct : monoclonal antibodies (MoAbs) are pre-conjugated to fluorochromes
2. Indirect : Unconjugated MoAbs

Question 15

What are 2 common data display?

Answer 15

1. histogram

2. dot plot

Question 16

What dye is used for analysis of DNA?

Answer 16

Propidium iodide(PI):
undergoes a dramatic increase in fluorescence upon binding DNA.
it requires the plasma membrane to be permeable.

Question 17

What is the excitation and emission value for propidium iodide (PI)?

Answer 17

• excitation = 488nm

- emission = 620nm

Question 18

How can you measure cell viability(healthy cells)?

Answer 18

• PI cannot normally cross the cell membrane
• if the PI penetrates the cell membrane it is assumed to be damaged.
• when PI enters cell it binds to DNA causing bright fluorescent, indicates cells are damaged or dead.

Question 19

How do we measure apoptosis?

Answer 19

1. by staining the cells with the PI dye and Annexin receptors with Fluorchrome
2. When cell is not damaged the phosphatidyl serine is intact on the inside of cell so annexin receptors can’t bind , no colour (double negative)
3. Early apoptosis phosphotidyl serine flippped on outside so annexin can bind emitting green fluorichrome (single positive for FITC, bottom right)
   4 late apoptosis damage to cell membrane makes it permeable to PI which enters the cell and emits red flurochrome (double positive fitc an PI)

Question 20

What is the issue with using sub-G0 peak to measure apoptosis(using PI dye)?

Answer 20

1. unclear whether sub-G0 peak is formed due to apoptosis or DNA fragment
2. not all cell type show sub-G0 peak when they apoptose.
   => to overcome this we use annexinV-FITC with PI

Question 21

What is 7- aminoactinomycine D (7AAD)?

Answer 21

7-aminoactinomycine D (7AAD)

• excitation = 488nm
• emission = 660nm
• DNA specific : intercalates in G-C regions
• long emission wavelength

Question 22

How does cell sorting work?

Answer 22

-> physical separation of defined cell populations without cultivation.

Question 23

What are applications of flow cytometry?

Answer 23

• immunophenotyping of leukaemias and lymphomas
• detection of MRD
• stem cell enumeration
• CD4/CD8 in HIV
• measurement of intracellular cytokines
• study of cell cycle , viability and apoptosis
• measurement of cell proliferation
• assessment of transfection efficiency.

Question 24

What is the difference between flow cytometry and flow sorting?

Answer 24

Flow cytometry: measuring properties of cell in flow

Flow sorting : separating cell based on properties measured in flow (also called FACS)