flow cytometry Flashcards
What is flow cytometry?
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
What’s the difference between flow cytometry and flow sorting?
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)
What can a flow cytometer tell us about a cell?
3
1) its relative size
2) its relative granularity/internal complexity
3) its relative fluorescence intensity
What can the relative fluorescent intensity be used to look for?
(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)
What are some ways to visualise fluorescent cells?
- fluorescence microscopy
- flow cytometry
+ of flow cytometer over microscope?
- how many cells can be viewed at one time?
- cell type viewed
- quantitative?
- accuracy
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)
What are the major components of a flow cytometry machine?
fluidics
optics
electronics
flow cytometry machine
expand on fluidics
Where cells in suspension flow in a single file
flow cytometry machine
expand on optics
The cells flow through an illuminated volume where the laser hits the cell and the cells scatter light and emit fluorescence
flow cytometry machine
expand on electronics
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
Fluidics- flow cytometry machine
describe the flow of cells in the machine and how this is achieved
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.
what is meant by hydrodynamic focusing
The introduction of a large volume into a small volume
Describe lasers as the light source in the optics part of a flow cytometer
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)
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?
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
flow cytometry
what is forward light scatter proportional to
size of the cell
flow cytometry
what is 90° light scatter proportional to
granularity of the cell
describe the Channel layout for laser-based flow cytometry
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.
Describe the electronics part of flow cytometry, what happens in this part
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
What is Stokes Shift?
where can this data be represented?
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)
what creates the difference between the 2 peaks seen in stokes shift?
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)
what are the 3 common fluorochromes
FITC
fluorescein isothiocyanate
PE
phycoerythrin
PerCP
peridinin chlorophyll protein
fluorochrome: FITC
wavelength they emit?
colour they emit?
520 nm
green
fluorochrome: PE
wavelength they emit?
colour they emit?
580 nm
orange
fluorochrome: PerCP
wavelength they emit?
colour they emit?
620 nm
red
why can all 3 fluorochromes be used at the same time?
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
What are some examples of samples of single cells in suspension?
- peripheral blood
- bone marrow
- fine needle aspirate
- CSF and other fluids
- fresh tissue
What are the two methods of labelling in immunofluorescence?
direct
indirect
methods of labelling in immunofluorescence
describe the direct method
Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes
methods of labelling in immunofluorescence
describe the indirect method
Unconjugated MoAb (XMoAb labelled with F-Ab) (labelled antibody w/fluorochrome on top of unlabelled antibody)
what is the difference between the data presented on a dot plot and data on a histogram?
dot plot = looks at 2 parameters at the same time
histogram = looks at 1 parameter
what data does a dot plot tell you
look at 2 parameters at the same time
forward scatter (cell size) vs side scatter (granularity of cell)
what data does a histogram tell you (x and y axis)
X axis = fluorescence intensity
Y axis = number of cells
Tells you how many cells there are at each fluorescence intensity
what is the difference between gating and analysis and what they can be used for
GATING can be used to look at specific populations
ANALYSIS can be used to quantitate anything
Using 3 different fluorochromes at a time will give you the ability to distinguish between X different populations
what is X
Using 3 different fluorochromes at a time will give you the ability to distinguish between 8 different populations
Describe one of the applications of flow cytometry.
analysis of cell cycle position by quantitation of cellular DNA
fast, accurate determination of cell cycle distributions
how is PI used to analyse the cell cycle
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
how can PI be used to check the viability of a cell
PI cannot normally cross the membrane
if the PI penetrates the membrane = fluorescence then it is assumed that the cell is damaged/dead
What is apoptosis, and what are some of its characteristics?
3
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
Describe the different steps between necrosis and apoptosis
3
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
what can flow cytometry be used to quantitate in the apoptotic pathway
caspase levels
What are some detection methods for apoptosis?
- staining with PI dye
(cells fixed) - PTS (phospatidyl serine) detection
(cells not fixed) - staining with 7-aminoactinomycin D
(cells not fixed)
detection methods for apoptosis
expand on PTS method
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
PTS method, what differences would be seen in early apoptosis compared to late apoptosis
describe these differences
early apoptosis:
- positive for annexin
- negative for PI
late apoptosis:
- positive for annexin
- positive for PI
(as membrane has disintegrated)
detection methods for apoptosis
+ of staining with 7-AAD
DNA specific
only uses one dye
long wavelength = flexible use
PI combined with AnnexinV-FITC
state what is seen in live cells
- 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)
PI combined with AnnexinV-FITC
state what is seen in early apoptotic cells
- 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
PI combined with AnnexinV-FITC
state what is seen in late apoptotic/necrotic cells
- 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
List some applications of flow cytometry
- 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
FLOW SORTING
how is flow sorting different to flow cytometry
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.
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
- Cell cultures
- Molecular biology
- Biochemistry
- Microscopy
