flow cytometry

Question 1

What is flow cytometry?

Answer 1

It’s a technique which simultaneously measures several physical characteristics belonging to
a SINGLE CELL in SUSPENSION.

This is done by LIGHT SCATTER and FLUORESCENCE

Question 2

What’s the difference between flow cytometry and flow sorting?

Answer 2

FLOW CYTOMETRY: - measuring properties of cells in flow

FLOW SORTING:

• sorting (separating) cells based on properties measured in flow
• also called Fluorescence-Activated Cell Sorting (FACS)

Question 3

What can a flow cytometer tell us about a cell?

3

Answer 3

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

Question 4

What can the relative fluorescent intensity be used to look for?
(4)

Answer 4

It can be used to look at different characteristics of the cell, such as:

• cell surface receptors and antigens
• adhesion molecules
• levels of intracellular cytokines and enzymes
• DNA (can look at cell cycle, etc)

Question 5

What are some ways to visualise fluorescent cells?

Answer 5

• fluorescence microscopy

- flow cytometry

Question 6

+ of flow cytometer over microscope?

• how many cells can be viewed at one time?
• cell type viewed
• quantitative?
• accuracy

Answer 6

flow cytometry:
- Can look at thousands of cells at a time, very quick

• Can look for rare cells
• Very quantitative, looks at thousands of cells
• Very accurate fluorescence intensity

Fluorescence microscope:

• Can only look at a limited number of cells in each field down a microscope, time consuming
• Cannot look for rare cells easily, will have to look at thousands of fields
• Not very quantitative, done by eye, around 20 cells per field
• Intensity variable (inaccurate)

Question 7

What are the major components of a flow cytometry machine?

Answer 7

fluidics

optics

electronics

Question 8

flow cytometry machine

expand on fluidics

Answer 8

Where cells in suspension flow in a single file

Question 9

flow cytometry machine

expand on optics

Answer 9

The cells flow through an illuminated volume where the laser hits the cell and the cells scatter light and emit fluorescence

Question 10

flow cytometry machine

expand on electronics

Answer 10

The fluorescence is collected, filtered and converted to digital values that are stored on a computer

Analogue —> digital —> data which can be analysed on computer

Question 11

Fluidics- flow cytometry machine

describe the flow of cells in the machine and how this is achieved

Answer 11

You need to have the cells in suspension flow in single file.

This is accomplished by injecting a sample into a sheath fluid as it passes through a small (50-300 µm) orifice
The sample fluid flows in a central core that does not mix with the sheath fluid - making it laminar flow.

The introduction of a large volume into a small volume - this is called hydrodynamic focusing.

Question 12

what is meant by hydrodynamic focusing

Answer 12

The introduction of a large volume into a small volume

Question 13

Describe lasers as the light source in the optics part of a flow cytometer

Answer 13

It is a single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths

• it can provide from milliwatts to watts of light
• can be inexpensive, air-cooled units or expensive, water-cooled units
• they provide coherent light (single frequency)

Question 14

How do we get information from the way light scatters when it hits the cell?

• what are the 2 different ways light can scatter?
• what do each of these scatters represent?

Answer 14

When the light hits the cell and it scatters, it scatters in two directions:

• forward light scatter, which is proportional to the size of the cell
• 90° light scatter (side scatter), which is proportional to the granularity of the cell

Question 15

flow cytometry

what is forward light scatter proportional to

Answer 15

size of the cell

Question 16

flow cytometry

what is 90° light scatter proportional to

Answer 16

granularity of the cell

Question 17

describe the Channel layout for laser-based flow cytometry

Answer 17

Laser hits the cells flowing in single file.

Cells have been labelled with 4 different antibodies with 4 different colours on them.

Fluorescence is emitted from the cells and it is picked up by a photo multiplier tube (PMT) after the light has gone through filters and mirrors.

At the PMT the light is converted from analogue –> digital.

Question 18

Describe the electronics part of flow cytometry, what happens in this part

Answer 18

It is where the processing of signals from detectors takes place.
It is the analog-digital conversion so the data can be analysed on a computer

Question 19

What is Stokes Shift?

where can this data be represented?

Answer 19

Stokes Shift is the energy difference between the lowest energy peak of absorbance and the highest energy of emission.

represented on FITC (emission spectrum of fluorochrome)

Question 20

what creates the difference between the 2 peaks seen in stokes shift?

Answer 20

Fluorescence happens when a fluorochrome is excited by a laser –> goes back to unexcited state and emits fluorescence at a higher wavelength. The difference between these 2 peaks is called stokes shift.
FITC excited by laser –> emits at a longer wavelength of 520nm (green)

Question 21

what are the 3 common fluorochromes

Answer 21

FITC
fluorescein isothiocyanate

PE
phycoerythrin

PerCP
peridinin chlorophyll protein

Question 22

fluorochrome: FITC

wavelength they emit?

colour they emit?

Answer 22

520 nm

green

Question 23

fluorochrome: PE

wavelength they emit?

colour they emit?

Answer 23

580 nm

orange

Question 24

fluorochrome: PerCP

wavelength they emit?

colour they emit?

Answer 24

620 nm

red

Question 25

why can all 3 fluorochromes be used at the same time?

Answer 25

Because all of these emit at different wavelengths, we can use them all at the same time 3 different antibodies with 3 different fluorochromes on them at the SAME time and get different information from them all

Question 26

What are some examples of samples of single cells in suspension?

Answer 26

- peripheral blood  - bone marrow  - fine needle aspirate  - CSF and other fluids  - fresh tissue

Question 27

What are the two methods of labelling in immunofluorescence?

Answer 27

direct indirect

Question 28

methods of labelling in immunofluorescence describe the direct method

Answer 28

Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes

Question 29

methods of labelling in immunofluorescence describe the indirect method

Answer 29

``` Unconjugated MoAb (XMoAb labelled with F-Ab) (labelled antibody w/fluorochrome on top of unlabelled antibody) ```

Question 30

what is the difference between the data presented on a dot plot and data on a histogram?

Answer 30

dot plot = looks at 2 parameters at the same time histogram = looks at 1 parameter

Question 31

what data does a dot plot tell you

Answer 31

look at 2 parameters at the same time forward scatter (cell size) vs side scatter (granularity of cell)

Question 32

what data does a histogram tell you (x and y axis)

Answer 32

X axis = fluorescence intensity Y axis = number of cells Tells you how many cells there are at each fluorescence intensity

Question 33

what is the difference between gating and analysis and what they can be used for

Answer 33

GATING can be used to look at specific populations ANALYSIS can be used to quantitate anything

Question 34

Using 3 different fluorochromes at a time will give you the ability to distinguish between X different populations what is X

Answer 34

Using 3 different fluorochromes at a time will give you the ability to distinguish between 8 different populations

Question 35

Describe one of the applications of flow cytometry.

Answer 35

analysis of cell cycle position by quantitation of cellular DNA fast, accurate determination of cell cycle distributions

Question 36

how is PI used to analyse the cell cycle

Answer 36

PI (propidium iodide) is a fluorescent dye that preferentially binds to DNA holes punched in cell membrane, PI enters cell and can bind to DNA and histogram can be used to show how many cells there are at each stage of the cell cycle by the fluorescence

Question 37

how can PI be used to check the viability of a cell

Answer 37

PI cannot normally cross the membrane if the PI penetrates the membrane = fluorescence then it is assumed that the cell is damaged/dead

Question 38

What is apoptosis, and what are some of its characteristics? | 3

Answer 38

Apoptosis is programmed cell death where the cell goes through a highly regulated process of “dying”. Characteristics are:  - condensation of the chromatin material  - blebbing of nuclear material  - degradation of DNA giving rise to distinctive 'ladder' pattern on DNA gel electrophoresis

Question 39

Describe the different steps between necrosis and apoptosis | 3

Answer 39

NECROSIS: - small blebs form; the structure of the nucleus changes  - the blebs fuse and become larger; no organelles are located in the blebs  - the cell membrane ruptures and release the cell's contents; the organelles are not functional  APOPTOSIS:  - small blebs form - the nucleus begins to break apart, and the DNA breaks into small pieces; the organelles are also located in the blebs  - the cell breaks into several apoptotic bodies; the organelles are still functional

Question 40

what can flow cytometry be used to quantitate in the apoptotic pathway

Answer 40

caspase levels

Question 41

What are some detection methods for apoptosis?

Answer 41

- staining with PI dye (cells fixed) - PTS (phospatidyl serine) detection (cells not fixed) - staining with 7-aminoactinomycin D (cells not fixed)

Question 42

detection methods for apoptosis expand on PTS method

Answer 42

phosphatidyl serine, can be detected by incubating the cells with fluorescein-labeled Annexin V, and PI (cells not fixed)  Note: Phosphatidyl serine is something that is usually found INSIDE cell but after apoptosis it flips to the OUTSIDE of the membrane

Question 43

PTS method, what differences would be seen in early apoptosis compared to late apoptosis describe these differences

Answer 43

early apoptosis: - positive for annexin - negative for PI late apoptosis: - positive for annexin - positive for PI (as membrane has disintegrated)

Question 44

detection methods for apoptosis + of staining with 7-AAD

Answer 44

DNA specific only uses one dye long wavelength = flexible use

Question 45

PI combined with AnnexinV-FITC state what is seen in live cells

Answer 45

* Phosphatidyl serine is found inside the cell * Live cells are negative for PI and AnnexinV-FITC because Phosphatidyl serine is not on the outside so AnnexinV-FITC cannot bind and PI cannot enter the cell because the membrane is intact (not damaged)

Question 46

PI combined with AnnexinV-FITC state what is seen in early apoptotic cells

Answer 46

* Phosphatidyl serine moves to the outside of the cell * Cell is found positive for AnnexinV-FITC because it can now bind to the phosphatidyl serine on the outside of the cell * Cell is still found negative for PI because the membrane is still intact and the PI cannot move inside the cell

Question 47

PI combined with AnnexinV-FITC state what is seen in late apoptotic/necrotic cells

Answer 47

* Cell is found positive for PI because the cell is damaged at this point and the PI can move into the cell * Cell is positive for both PI and AnnexinV-FITC

Question 48

List some applications of flow cytometry

Answer 48

- 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 49

FLOW SORTING how is flow sorting different to flow cytometry

Answer 49

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 50

As a result of flow sorting, we get a sample of very purified cells, with endless possibilities in research. give some examples of where these purified cells can be used

Answer 50

* Cell cultures * Molecular biology * Biochemistry * Microscopy