Intro to Flow Cytometry Flashcards by No Na

What is flow cytometry?

This is a technique which simultaneously measures several physical characteristics belonging to a single cell in suspension

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How are cells held in suspension and measured for flow cytometry?

This is done by light scatter and fluorescence

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What is the function of flow cytometry?

Flow Cytometry: Measuring properties of cells in flow

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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)

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What information do we gain from a flow cytometer?

Its Relative Size
Its Relative Granularity/Internal Complexity
Its Relative Fluorescence Intensity

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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

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What are the ways of visualising flow cytometer results?

Fluorescence Microscopy

- Flow Cytometry

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Why is flow cytometery preferred over fluorescence microscopy?

Flow cytometer is quick
Quantitative
Can find rare cells

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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

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What instrument is used to interrogate cells?

analyser used to interrogate cells

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What are the 3 compartments of the analysing machine?

The way the machine works is broken down into 3 compartments:

Fluidics
Optics
Electronics

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Describe fluidics component of the analyser?

Fluidics

Cells in suspension
Flow in single-file through

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What are the optics in an analyser?

Optics

an illuminated volume
where they scatter light and emit fluorescence
is collected, filtered

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Describe the electronic part of the analyser

Electronics

converted to digital values
that are stored on a computer

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How are cells required to be arranged in an analyser?

Need to have cells in suspension flow in single file

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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

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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

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What is hydrodynamic focusing?

Introduction of a large volume into a small volume

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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