Intro to Flow Cytometry Flashcards
What is flow cytometry?
This is a technique which simultaneously measures several physical characteristics belonging to a single cell in suspension
How are cells held in suspension and measured for flow cytometry?
This is done by light scatter and fluorescence
What is the function of flow cytometry?
Flow Cytometry: Measuring properties of cells in flow
What is the role of flow sorting?
Flow Sorting: Sorting (separating) cells based on properties measured in flow
Also called Fluorescence-Activated Cell Sorting (FACS)
What information do we gain from a flow cytometer?
- Its Relative Size
- Its Relative Granularity/Internal Complexity
- Its Relative Fluorescence Intensity
What aspects of a cell can be measured using a flow cytometer?
Can measure anything on cell surface ie.
- receptors
- adhesion molecules etc.
- cytokines
- enzymes in cytoplasm
Can also interrogate cell DNA; cell cycle, cell viability & apoptosis
What are the ways of visualising flow cytometer results?
- Fluorescence Microscopy
- Flow Cytometry
Why is flow cytometery preferred over fluorescence microscopy?
- Flow cytometer is quick
- Quantitative
- Can find rare cells
Why is fluorescence microscopy not a preferred method?
Can only view limited no. of cells in field view looking down a microscope
To find rare cells, need to interrogate thousands of field views
Microscopy not very quantitative as carried out by eye - subjective
Fluorescence varies in microscope marking - subjective
What instrument is used to interrogate cells?
analyser used to interrogate cells
What are the 3 compartments of the analysing machine?
The way the machine works is broken down into 3 compartments:
- Fluidics
- Optics
- Electronics
Describe fluidics component of the analyser?
Fluidics
- Cells in suspension
- Flow in single-file through
What are the optics in an analyser?
Optics
- an illuminated volume
- where they scatter light and emit fluorescence
- is collected, filtered
Describe the electronic part of the analyser
Electronics
- converted to digital values
- that are stored on a computer
How are cells required to be arranged in an analyser?
Need to have cells in suspension flow in single file
How are cells kept in single file?
Accomplished by injecting sample into a sheath fluid as it passes through a small (50-300 µm) orifice
Where in the analyser does the sample fluid flow?
Sample fluid flows in a central core that does not mix with the sheath fluid - Laminar flow
What is hydrodynamic focusing?
Introduction of a large volume into a small volume
Describe the flow of cells through the analyser
- Cells flow through nozzle tip (small orifice)
- forced to flow in single file due to sheath fluid flowing
around sides: hydrodynamic focussing - Cells need to be single file for when laser hits
- Laser hits, light scatters and picked up by the detectors
What is a laser?
Single wavelength of light (a laser line) or (more rarely) a mixture of wavelengths
light source
Describe the features of lasers
- 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 is the benefit of using laser as the light source?
Even without fluorescence and antibody marking, lasers provide a lot of info about the cells
How does the laser provide cellular info?
Laser hits the cell and scatters light into 2 different directions:
- forward scatter
- side scatter
What does forward light scatter tell us?
Forward light scatter = proportional to size of the cell
What info do we gain from side scatter?
Side scatter 90° = proportional to granularity / internal complexity of the cell
What does a white cell dot plot show us?
Every dot represents ‘an event’ (cell)
The screen is refreshed every second to show 1000s of cells
Just on basis of forwards and side scatter we can see distinctions between populations
What do the axis on a white cell dot plot show?
X axis - increase in forward scatter
Y axis - increase in side scatter
Describe the basic pattern of peripheral blood seen on a white cell dot plot
RBCs and debris in bottom left corner
lymphocytes v. small and not very granular
monocytes are bigger and granular
a large pop of neutrophils
Describe laser-based flow cytometry
Cells labelled with fluorescently labelled antibodies
Lasers hit cells emerging from nozzle tip
Light emitted from cells and picked up by PMTs after light has travelled through mirrors and filters
What are electronics of flow cytometry?
Processing of signals from detectors
Analog-Digital Conversion
When does fluorescence occur?
Fluorescence occurs when fluorochrome is excited by a laser and returns to unexcited state via emission
⇒ emits fluorescence at a higher wavelength
What is stokes shift?
Stokes Shift is the energy difference between the lowest energy peak of absorbance and the highest energy of emission
Give examples of common fluorochromes and dyes
Green: Fluorescein isothiocyanate (FITC)
Orange: Phycoerythrin (PE)
Red: Peridinin Chlorophyll Protein (PerCP)
How can we investigate different parameters at once?
As these fluorochromes emit at different wavelengths we can detect them all at the same time to investigate 3 different parameters at once
What is the purpose of filters and mirrors in flow cytometry?
The broad emission spectra overlap which is why we require filters and mirrors to separate them out
Give examples of good samples to use in flow cytometry
The following are good samples to use in a flow cytometer:
- Peripheral blood
- Bone marrow
- Fine Needle Aspirate
- CSF and other fluids
- Fresh Tissue
What are the 2 immunofluorescence labelling methods?
- Direct
- Indirect
What is the direct method of immunofluorescence?
Monoclonal antibodies (MoAbs) are pre conjugated to fluorochromes (simplest and cleanest way)
Describe the indirect way of immunofluorescence labelling
Unconjugated MoAbs, require secondary Abs with flurocohrome
How can we display the data collected from flow cytometry?
> 2 ways of displaying data
- Dot plot
- Histogram
What is the use of dot plots?
Dot plot allows the visualization of 2 parameters at the same time
(forward and side scatter)
How are histograms used?
Histogram only looks at one parameter
fluorescence intensity
What info do dot plots show us?
Using 2D dot plot can identify 4 populations of cells:
- Double -ve for both
- Single +ve for FITC
- Single +ve for PE
- Double +ve for both
Can quantitate cells
What is gating?
A way of manipulating data and displaying it to analyse it
What is a common use of gating?
Drawing a region around lymphocytes and using CPU visualised those specific cells on basis of their 2 fluorochromes (CD3 FITC and CD4 PE)
How do we analyse flow cytometry data?
Put markers on histograms and CPU machines quantitate the no. of cells
Draw quadrants to analyse proportion of cells in each quadrant
How does the no. of fluorochromes used enable broader data range?
By increasing no. of fluorochromes to 3 we can identify up to 8 populations:
-ve for all 3
+ve for all 3
Or combinations in between
By increasing colours we can increase populations to analyse
