Tut 7- Flow Cytometry Flashcards
What is flow cytometry?
Flow = working in a fluid stream
Cytometry = the study of cells
So flow cytometry is the study of cells in a fluid stream
What does flow cytometry do?
Measures and counts every single cell in a fluid stream and the
Relative size (Forward Scatter – FSC)
Relative granularity or internal complexity (Side Scatter – SSC)
Amount of fluorescence i.e., detects the expression of molecules in or on the cell using fluorochrome-conjugated monoclonal antibodies (eg surface receptors)
Rapid and sensitive (speeds of 2,000 - 10,000 cells per second)
What are some advantages of flow cytometry?
Multi-parameter analysis (1-8) i.e., multiple fluorescent colours to differentiate between different cell markers
Rapid analysis (2-10,000 cells/s)
Significant number of cells sampled
Sensitive identification of rare events (eg Minimal Residual Disease detection)
What are some disadvantages of flow cytometry?
Single-cell suspensions only
No tissue context i.e., location in a tissue (as seen by microscopy)
Unable to detect intracellular location
Cells cannot be recovered from instrument (unless using a cell sorter)
What can you analyse with flow cytometry?
Wide range of fluorescently labelled “markers” to target cell membrane, proteins, and nucleic acid
Immunophenotyping of cell populations & cell counts
Viability, apoptosis, cell cycle and proliferation
Cell function: phagocytosis, ROS, cytokine release
Direct detection of bacteria, fungi, parasite or virus
Monitoring infections and antimicrobial therapy
Detection of non-culturable organisms
Cytometry has 3 components, describe them
Fluidics which introduce and focus cells in front of the lasers
Optics to generate and collect the fluorescent light signals
Electronics to convert light signals to proportional digital signals
Describe fluidics in more detail
Fluidics introduce (or carry) cells into the instrument and focus them in front of the laser /s. Cells must flow in single file to allow accurate analysis of each cell. Sample is injected under pressure into the sheath fluid and in most instruments undergoes a process known as “Hydrodynamic focusing” (the Attune flow cytometers use acoustic focusing). When laminar flow is achieved, the sample fluid flows in a central core and does not mix with the outer sheath fluid.
A flow cytometer has a light source (laser) and optics. Describe these.
Excitation optics (lasers)
A laser is used to provide a single wavelength of light.
Multiple lasers can be installed to provide coincident light
of different wavelengths.
The laser light beam is shaped by excitation lenses to focus on cells in fluid stream.
Collection optics (detectors)
Emitted light is directed to appropriate detectors by lenses and filters.
Light from forward scatter, side scatter and emitted fluorescence are detected.
Describe Optics: Forward Scatter (FSC)
Forward scatter is the amount of light diffracted or scattered at narrow angles to the axis of the laser beam.
FSC is related to cell size and surface area but it is NOT a direct measure of cell diameter or circumference.
When light reaches the FSC detector, it generates a voltage pulse signal which is proportional to the amount of light received.
Describe Optics: Side Scatter (SSC)
Side scatter is the amount of light reflected or diffracted at 90o to the axis of the laser beam.
SSC is related to cell granularity and complexity.
Light is focused through a lens and is collected by a detector usually located 90o from the laser’s path.
Why do yo add fluorescence to cytometry?
Relative size and complexity are not always sufficient to discriminate between populations. For example B lymphocytes, T lymphocytes and natural killer cells are all similar in size and complexity. The addition of antibodies directed at cell surface markers unique to each of these populations, labelled with different fluorophores (or colours) provides another parameter we can use to determine these populations.
What is a fluorophore?
It’s a molecule which:
Absorbs energy of a specific wavelength and
Emits unused energy at a different (longer) wavelength of light
Define multiparameter analysis
Multiple markers can be analysed by choosing 800
fluorophores with different excitation wavelengths and/or emission profiles.
What are some information notes about electronics of flow cytometry?
Convert the collected light signal into a number we can quantify and analyse statistically.
As a cell passes in front of the laser it creates a voltage pulse, converting the light signal into a digital signal.
Analysing the voltage pulse provides information on the cell.
Data (FSC, SSC, fluorescence) is recorded for every ‘event’, i.e., cell or particle, that passes through the laser interrogation point.
What does the height, area and width refer to with flow cytometry?
Height = Maximum signal measured
Area = Total fluorescence of cell
Width = Time taken to pass through laser
What are the 4 main steps for flow cytometry?
- Fluorophores are excited by laser
- FSC, SSC and Fluorescence collected
- Conversion of scattered and fluorescent light to digital pulse
- Data for each event is plotted in dotplots and histograms
Describe cytokine bead array
A. The microspheres or ‘beads’ contain unique ratios of two red dyes eg red and near infra-red (NIR)
B. Antibodies to different cytokines are coupled on each bead eg A5=anti-IL6
C. Beads are incubated with sample, followed by a fluorescently conjugated
detection antibody eg anti-IL6-PE
D. A panel of beads are selected for the cytokines of interest
E. A standard curve is prepared for each cytokine
F. Samples are analysed and the mean fluorescence intensity (MFI) compared to
the standard curve to calculate the concentration
What are the principles of phagocytosis essay?
A. Incubate macrophages with fluorescent E. coli
B. Macrophages will phagocytose the bacteria. The number of bacteria phagocytosed will vary depending on the incubation time and whether the bacteria are opsonised or unopsonised.
C. Analyse by flow cytometry
Define viral detection and influenza “typing”
Antibodies on the microsphere surface
capture influenza viruses present in patient
samples. Fluorescent influenza specific
polyclonal antibodies are then applied
which bind to the captured influenza virus
particles and the fluorescent signal on each
bead analysed by flow cytometry.
Covid 19- FCM analysis findings
increased total neutrophils
reduced total lymphocytes
increased serum levels of IL-6 and
of C-reactive protein
reports of abnormally high plasma
levels of innate cytokines (eg MCP-1, TNFα) or of high levels of proinflammatory cytokines (eg IL- 2, IL-7, IL-10, G-CSF)
Emerging data from elderly patients with Covid-19 in Modena, Italy show low T cells count, an increase in naïve helper T cells and a decrease in memory helper T cells
