Flow cytometry I - introduction Flashcards
What is flow cytometry?
Technique which simultaneously measures several physical characteristics belonging to a single cell in suspension by:
- Light scatter and fluorescence
What is flow sorting?
Sorting cells based on properties measured in flow
also called Fluorescence-Activated Cell Sorting (FACS)
What can a flow cytometer tell us about a cell?
- its relative size
- its relative fluorescence intensity
- its relative granularity
How does a flow cytometer get information about a cell?
It measures:
- Anything on the cell surface (receptors, adhesion molecules)
- intracellular cytokines or enzymes
- the DNA (cell cycle, apoptosis, viability of the cell)
What is an other method of cell visualisation than flow cytometry
Fluorescence microscopy
Difference between flow cytometer and fluorescent microscope
Fluorescent microscopy:
- only a limited view of cells - - hard to quantitate cells accurately (especially with the eye)
Flow cytometry:
-we are looking at thousands of cells every second
- the intensity of the fluorescence is quite variable and we make subjective judgements, whereas the flow cytometer it gives a very accurate view of how intense the fluorescence is
Advantages of flow cytometry
· quick
· can look for rare cells
· quantifiable
· more accurate and quantifiable intensity
Basics of Flow Cytometry Machine
· 3 components:
Fluidics
Optics
Electronics
Requirements in fluidics
Cells need to be in suspension to flow in a single file
How are the cells in suspension organised to flow in a single file?
Hydrodynamic Focusing
- injecting a sample fluid (cells) into central core
- this runs side by side to the sheath fluid (but they don’t mix), creating a Laminar flow
- sample cells pass through a small 50-300um orifice one by one at the junction where the analysis by lasers begins
What happens after cells flow in a single file?
Optics
-the laser hits the cells one by one
-light is scattered in two directions which are picked up by detectors, obtaining cellular information (without even adding any antibodies or fluorescence yet)
Forward light scatter → proportional to the size of the cell
90° light scatter (side scatter)→ proportional to the granularity/internal complexity of the cell
Features of lasers used in flow cytometry
- 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
What happens when laser light hits the cell
what is the wavelength of the laser light
- Light hits the cell and is scattered in a forward direction proportional to the size of the cell = forward light scatter
- Light is also scattered at a right angle to the cell proportional to granularity = side scatter
488nm
What happens after light scatters are produced?
The different cell populations already distinguished are labelled with different antibodies with fluorochromes on them (immunofluorescence)
Laser→ fluorescence is emitted from cells, passes through filters and mirrors and we pick that up by the photo multiplier tubes (PMTs)
Why are filters and mirrors needed?
We need the system of filters and mirrors because of the overlap in the emission spectrum of the different fluorochromes.
Mirrors are responsible for directing light through the detection path
Filters are placed in front of detectors to restrict light introduced to each PMT so that each detector is dedicated to measure fluorescence from a specific fluorochrome
Role of photo multiplier tubes (PMTs)
Electronics
-convert the light (analogue) into digital
How does fluorescence happen in flow cytometry?
Fluorescence happens when a fluorochrome is excited by a laser and then it goes back to its unexcited state, emitting fluorescence at a higher wavelength
What is the Stokes shift?
The Stokes shift is the energy difference between the peak excitation and peak emission wavelengths.
Fluorochromes and dyes commonly used in immunofluorescence
· Fluorescein isothiocyanate (FITC)→ emits green
· Phycoerythrin (PE)→emits orange
· Peridinin Chlorophyll Protein (PerCP)→emits red
*all excited by the common 488nm laser
Why are FITC, PE and PerCP commonly used together in immunofluorescence?
Because they emit at different wavelengths we can detect them all at the same time. We can add in three different antibodies with different fluorochrome on them all at the same time to obtain information on the cells.
Single cells in suspension for flow cytometry include:
Peripheral blood Bone marrow Fine needle aspirate CSF and other fluids Fresh tissue
How do we label the cell with antibodies?
Direct Labelling
- monoclonal antibodies (MoAbs) are preconjugated to fluorochromes and then added to cell suspension
- wash and then put through flow cytometer
Indirect Labelling
- unconjugated MoAbs added to cell suspension
- wash
- then we add a secondary antibody with a fluorochrome on it to the primary antibody
- wash and then put through flow cytometer
How to display data from a flow cytometer on a computer
Two ways of displaying data on the computer:
- Dot plot (looks at two parameters→e.g. forward scatter & side scatter)
- Histogram (looks at one parameter)
How many populations can you identify with two fluorochromes?
Using our two-dimensional dot plot, we can identify four populations of cells, either:
- negative for both
- single positive for FITC
- single positive for PE
- double-positive for both
Principle of flow cytometry data analysis
GATING
-creating ‘gates’ on the dot plot to distinguish cells of interest and analyse those specific cells
Then, analysis is used to quantitate the populations.
How many populations can you identify with three fluorochromes?
If we increase that to three colours, we can now look at eight different populations of cells, either positive for all three, negative for all three or combinations in between. Therefore, three different fluorochromes will give you the ability to distinguish eight different populations.
