Flow Cytometry I + II Flashcards
Define flow cytometry
Measures properties in flow of single cells in suspension
Carried out using light scatter and fluorescence
Define flow sorting
Sorting (separating) cells based on properties in flow Also called FACS (fluorescence-activated cell sorting)
What can a flow cytometer tell us about a cell?
- Its relative size
- Its relative granularity/ Internal complexity
- Its relative fluorescence intensity
What can the relative fluorescent intensity be used for?
Can be used to look at different characteristics of the cell such as:
- Cell surface receptors
- Adhesion molecules
- Levels of intracellular cytokines and enzymes
- DNA
What are some of the ways in which we are able to visuallise fluorescent cells?
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Fluorescence microscopy
- Not very quantitative as it takes a long time to accurately quantitate the cells
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Flow cytometry
- Looking at thousands of cells, able to asses the intensity of each individual cell
Summarise the steps of a flow cytometer
Fluidics → Where cells in suspension flow single file
Optics → The cells flow through an illuminated volume where they scatter light and emit fluorescence
Electronics → The fluorescence is collected, filtered and converted into digital values which are stored on the computer
Describe fluidics in flow cytometry
- Cells will be introduced and forced out single file in suspension flow
- This is accomplished by injecting a sample into a sheath fluid as it passes through a small orifice
- A laser will hit the flow cells and light will be scattered due to cell size and granularity
- Or cells have been stained with fluorescent markers, the cells will also emit fluorescence
Define laminar flow and hydrodynamic focusing in terms of glow cytometry
Laminar flow = sample fluid flows in a central core that doesnt fix with sheath fluid
Hydrodynamic focusing = Introduction of a large volume into a small volume
Describe the optics part of the flow cytometer
Focus on laser
- 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/ expensive, water cooled units
- They provide coherant light (single frequency)
What happens when the laser hits a cell?
When the laser hits the cell light will be scattered in two directions
1) Light is scattered in a forward direction which is proportional to the size of the cell
2) Light will be scattered in a 90 degree angle which is proportional to the granularity of the cell
Describe the electronics part of flow cytometry
It is where light signals from detectors is converted into a digital signal
Describe the basis of fluorescence
When a fluorochrome is excited by a laser at a certain wavelength the light it emits following resumption back to its unexcited state is at a longer wavelength
There are two wavelengths
- Emission excitation wavelength
- Emission wavelength
What is Stokes Shift
Is the energy difference between the lowest energy peak of absorbance and the highest energy peak of emission
What is a fluorochrome and give examples of some common fluorochromes
A fluorochrome is a chemical which can absorb energy from an excitation source (laser beam) and emit photons at a longer wavelength (fluorescence), which is captured by optical detectors of the flow cytometer
Fluorescein isothiocyanate (FITC) GREEN
Phycoerythrin (PE) ORANGE
Peridinin Chlorophyll Protein (PerCP) RED
What are the two methods of labelling in immunofluorescence
1) Direct - monoclonal antibodies are preconjugated to fluorochromes
2) Indirect - unconjugated MoAbs are added (primary, then secondary) and fluorophores are added on top of this
How is data following a flow cytometer presented?
Histogram = Where you can only measure 1 parameter at a time
Dot Plot = able to quantitate on the basis of two parameters (e.g forward scatter and side scatter)
How is cellular DNA detected in flow cytometry (for example analysis of the cell cycle>
Cellular DNA is detected using fluorescent dye which preferentially binds to DNA
Propidium iodide is the most commonly used dye
- It undergoes a dramatic increase in fluorescence upon binding DNA.
- It requires permeabilization of the plasma membrane which occurs in late apoptosis (must be late apoptosis or dead cells)
Explain how propidium iodide works
- Normally propidium iodide cannot cross the membrane
- If propidium iodide penetrates the membrane it is assumed to be damaged
- Cells that are brightly fluorescent with PI are severly damaged or dead
What is apoptosis and describe some characteristics
Apoptosis = Programmed cell death where cell goes through a regulated process of dying
Characteristics are:
- Condensation of chromatin material
- Blebbing of nuclear materal
- Internucleosomal degradation of DNA giving rise to ladder pattern on DNA gel electrophoresis
What is the difference between apoptosis compared to necrosis?
What detection methods can be used for apoptosis?
- Staining dye with propidium iodide
- Phosphatidyl serine can be detected by incubating cells with fluorescein-labeled Annexin and propidium iodide
- Staning with 7-aminoactinomycin D
How can you distinguish different populations of cells using propidium iodide and annexin?
⇒ If the cell doesnt show active AnnexinV or PI - Live cell
⇒ If the cell shows AnnexinV but not PI - Early apoptotic cell
⇒ If the cell shows both AnnexinV and PI - Late apoptotic/necrotic cell
Describe 7-Aminoactinomycin D (7-AAD)
- DNA-specific (intercalates in G-C regions)
- Long emission wavelength
- We can use this dye in conjunction with other antibodies and fluorochromes
- Allows us to carry out FITC and PE labelled antibodies alongside for simulatenous evaluation of DNA content and 2 colour immunofluoresence using a 488nm common laser
- We can use this dye in conjunction with other antibodies and fluorochromes
Describe cell sorting
- Still follows the same principle as cytometry - the cells are in suspension single file, a laser is put on them and light is picked up and emitted telling a computer what type of cell it is
- In cell sorting we can draw a range around the type of cells we want so the computer will sort them out
- The nozzle tip will always be vibrating so that the stream will break into droplets
- If this droplet contains the desired cell a charge will be added to cells of choice
- The charged droplets containing the cell will be collected into tubes by being pulled towards a deflection plate
- The rest of the cells will be collected for waste
