Flow cytometry Flashcards
What is flow cytometry?
Technique which simultaneously measures several physical characteristics belonging to a SINGLE CELL in SUSPENSION
This is done by LIGHT SCATTER and FLUORESCENCE
What is 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?
- Its Relative Size
- Its Relative Granularity/Internal Complexity
- Its Relative Fluorescence Intensity
- In the most recent leukocyte typing workshop over 160 antibodies were classified into defined groups
- These antibodies bind to cytokines or chemokine receptors, sensory molecules, cytokine or chemokine ligands, metabolic proteins, adhesion molecules, enzymes, structural proteins and other important functional proteins within the cell.
- These antibodies can be used to identify specific cell subsets by their unique repertoire of molecular expression as well as their functional state using multiparameter flow cytometry.
What are the two methods of visualisation?
- Fluorescence Microsocopy
* Flow Cytometry
What are the basic of flow cytometry?
On image
Describe fluidics of cytometry
- Need to have cells in suspension flow in single file
- Accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice
- Sample fluid flows in a central core that does not mix with the sheath fluid - Laminar flow
- Introduction of a large volume into a small volume - Hydrodynamic Focusing
Describe the optical system
- 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)
Describe the light scatter phase
- This is the common 488 laser
- The laser hits the cell, and the cell scatters light in 2 directions
- Light scattered in the forward direction is proportional to the size of the cell
- Light scattered at a 90-degree angle is proportional to the granularity of the cell
What does a white cell population dot plot show?
- Shows peripheral blood without any fluorescence
- Called a dot plot
- Forward scatter x-axis, proportional to the size of the cell
- Granularity (side scatter) y-axis, 90o angle light scatter is proportional to granularity or internal complexity of cell
- Each dot represents a cell/event
- We know that the “blue” cells are lymphocytes as they have this size and granularity
Describe the principles of laser-based flow cytometry
- Just to show how the light goes through filters and mirrors and is then detected.
- Single wavelength laser used
- 4 fluorochromes used to label cells (chemical that can emit light upon excitation, in this case bonded to antibody), which emits light at different wavelengths (so each have different range of wavelength, and different colours)
- Filters and mirrors allows detection of fluorescence emitted by different fluorochromes independently
- Light signal in PMTs (photomultipliers) converted into digital signal in computer
Describe the computer phase
Electronics
• Processing of signals from detectors
o Analog-digital conversion
We can then do something with the digital data on our computers
Fluorescence
Stokes shift
Is the energy difference between the lowest energy peak of absorbance and the highest energy of emission
Describe the principle of immunofluorescence?
- Fluorescein isothiocyanate (FITC) GREEN
- Phycoerythrin (PE) ORANGE
- Peridinin Chlorophyll Protein (PerCP) RED
- PerCP at the top
- We can use these three colours together to measure 3 different parameters of the cell.
- There are overlaps in each of these spectrums So how do we differentiate between them?
- The filters and mirrors are used for this
- 4 colours come out, each detected by a different detector
- The laser light is going through the system of filters and mirrors so that by the time it reaches the detector, the detector only detects a narrow range of wavelength so we can differentiate between the 4 emissions from one laser.
- We label the cell with 4 different antibodies with 4 different colours on them with one laser line, so the cell is emitting signals depending on whether it is positive or negative for that antibody. We can restrict what the detectors are seeing in terms of wavelength
Give examples of the suspensions we can use
- Peripheral blood
- Bone marrow
- Fine Needle Aspirate
- CSF and other fluids
- Fresh Tissue
Describe the principles of direct and indirect immunofluorescence
Direct
• Monoclonal antibodies (MoAbs) are preconjugated to fluorochromes
Indirect
• Unconjugated MoAbs
• In the indirect method, the first stage is adding the primary antibody
• The second step is providing a secondary antibody to the primary antibody which has a fluorophore on
• Indirect method gives you an amplified sample (brighter signal) however there is a lot of background staining
What are the two ways of displaying data?
Histogram
• On the histogram, the x-axis fluorescent intensity, the y-axis cell count
• So, with a histogram, we can only measure one parameter at a time
• One dimensional
• E.g. x-axis: fluorescence intensity, y-axis: cell count
• Area under the histogram is the cell count
Dot plot
• With the dot plot, we can look at 2 parameters at a time and quantify cells accordingly
• Quantify 4 populations with 2 parameters, 8 populations with 3 parameters etc.
• Two dimensional
• E.g. y-axis: Forward scatter (size), x-axis: Side scatter (granularity/complexity)
• Each dot represents an event (cell)
