Practical 2: Flow Cytometry Flashcards
Define flow cytometry
A technique for counting, examining and sorting microscopic particles suspended in a stream of fluid
How does the flow cytometer work
A laser beam directed at a hydro-drynamically focused stream of fluid containing cells in single-file excites cells as they move by
A number of detectors pick measure properties such as forward scatter, side scatter and fluorescence at different wavelengths
The protein expression of cells can be measured by incubating cells with fluorescently labelled antibodies
How are fluorescently labelled antibodies used in flow cytometry?
The cell suspension is insubated with labelled antibody(s)
The cells are then aspirated from the sample tubes and hydrodynamically forced in single file by a laser
By combining antibodies with different fluorescent labels that emit light at different wavelengths we can carry out high throughout, multi-paramater analysis
The emitted light is detected by an optical system where specialised software on an adjoined computer can export the data as either dot plots or histograms
What is meant by hydrodrynamic focussing
Cells in sheath fluid flow through the tubing in a laminar fasion, as the tubing diameter narrows the cells are forced into single life before the laser
i.e. sheath fluid runs along either side of sample - so that sample is in laminar flow
Narrowing of tubing, along with work of sheath fluid, focuses the cells into single file
What are the two most common fluorophores using in flow cytometry?
Fluorescein (FITC)
R-phycoerythrin (PE)
What is spectral overlap?
One must be careful when deciding on which fluorescent label to use in order to avoid too much spectral overlap
This is where the light emitted by the fluorophores is of a similar wacelength and thus positive cell populations will bleed into each other e.g. there is significant overlap between FITC and PE
What does forward and side scatter measure?
Forward scatter - FSC
Side scatter - SSC
What is gating?
Gating is used to focus on a population of interest
- a method to define cell population of interest
- typically performed using forward and sde scatter
Within the lymphocyte gate what would CD3- CD56+ cells be?
NK cells
How many lasers are there in most flow cytometers?
There is usually 2, which can excit cells at different wavelengths
Three or more lasers can be used in larger machines
What does the light from the laser do?
The light interrogates cells as they pass by
NB: it is the light from the laser that does this not the laser itself
How are fluorophores used?
Light from a laser is absorbed by the fluorphores
The fluorophores cannot hold onto this light so it emits it back out
The light emitted is of a different wavelength to the light absorbed
This light is then detected by a detecter
How can flow cytometry be used on tissue?
A hydrodynamically focused beam of cells is required for flow cytometry
you cannot force solid material through the cytometer therfore tissue must be disaggregated/broken down
Enzymes are used to do this, a cell suspension is made
Need to be careful what enzyme you use though as you dont want it to target the antigen you are looking to detect in flow
Need to avoid weakening of antigen expression while disaggregating the tissue enough to make a cell suspension
What kind of controls do you use for flow?
You want to use negaive cells e.g. double negative cells, cells which contains neither signals you want to identify
How are mirrors used in flow cytometry?
Mirrors are used to change direction of light, it doesnt alter the wavelength of emitted light in anyway but they are a useful way of making our analysers small then they have to be, can make them a lot more compact through the use of mirrors
What is spectral overlap compensation?
The mathematical process by which we correct multiparameter flow cytometric data for spectral overlap
This overlap results from the use of fluorescent dyes that are measurable in more than one detector
This is the use of a mathematical algorithm to remove any “spilover” between two different fluorophore spectra
List the major advantages of Flow Cytometry
Generatioin of correlative information about single cells within a heterogenous sample preparation in a high-throughput fashion
Rapid a nd quanititative measurement of up to 30 parameters of cell phenotype simultaneously in a highly sensitive and reproducible manner
High speicificty for discrete cell subsets and rare populations such as IL-17A, IL-22 and IFN-y producing CD4+ cells in untreated psoriasis
Rare cell subsets with frequencies as low as 0.01% can be detected
Flow cytometric cell sorting also allows for the isolation (and susbequent investigation) of cells of interest to very high purity
What are the major limitations of flow cytometry?
Cells must be in a single-cell suspensioni to be evaluated -> problem for tissue as it must be disrupter which can affect antigens
The number of paramters per cell that can be mesured is limited by the number of detectors to less than two dozen but due to the spillover effect only 6-12 color experiments are routinely performed
There is a lack of standardisation on how flow data is analysed and reported
Flow data can become very complicated requiring very specialised human experts
