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Questions and Answers
What is FACS primarily used for?
What is FACS primarily used for?
FACS can sort up to 4 populations at the same time.
FACS can sort up to 4 populations at the same time.
True
Name one parameter measured by FACS.
Name one parameter measured by FACS.
Cell cycle
FACS is based on fluorescent-labeled _____ .
FACS is based on fluorescent-labeled _____ .
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Match the following cell separation methods with their descriptions:
Match the following cell separation methods with their descriptions:
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What parameter do photodiodes primarily detect?
What parameter do photodiodes primarily detect?
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Photomultiplier tubes (PMTs) do not provide any amplification of the signal inside.
Photomultiplier tubes (PMTs) do not provide any amplification of the signal inside.
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What is the function of sheath pressure in a cuvette?
What is the function of sheath pressure in a cuvette?
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The _______ separates the stream into single droplets during the drop formation process.
The _______ separates the stream into single droplets during the drop formation process.
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Match the components with their respective roles in the fluidics system:
Match the components with their respective roles in the fluidics system:
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In fluidics, what occurs after the droplets pass the laser?
In fluidics, what occurs after the droplets pass the laser?
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Histograms and dot plots can only be generated using a log scale.
Histograms and dot plots can only be generated using a log scale.
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What happens to the intensity of events detected in electronics?
What happens to the intensity of events detected in electronics?
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What does the nozzle size influence in a FACS experiment?
What does the nozzle size influence in a FACS experiment?
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Sheath pressure has no impact on stream stability in a FACS experiment.
Sheath pressure has no impact on stream stability in a FACS experiment.
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What is the main function of lasers in the optical system?
What is the main function of lasers in the optical system?
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In FACS, what must be considered for successfully sorting cells?
In FACS, what must be considered for successfully sorting cells?
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The output light from lasers is monochromatic and in phase.
The output light from lasers is monochromatic and in phase.
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The frequency in a FACS experiment determines the speed of __________.
The frequency in a FACS experiment determines the speed of __________.
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What do detectors do in the optical system?
What do detectors do in the optical system?
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The distribution of photons to detectors depends on the type of ______.
The distribution of photons to detectors depends on the type of ______.
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Which of the following is NOT a type of cell preparation method for FACS?
Which of the following is NOT a type of cell preparation method for FACS?
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Match the following cell types to their appropriate preparation methods:
Match the following cell types to their appropriate preparation methods:
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Match the components of the optical system with their functions:
Match the components of the optical system with their functions:
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Which statement best describes the light output of lasers?
Which statement best describes the light output of lasers?
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What should researchers consider when deciding how to collect sorted cells?
What should researchers consider when deciding how to collect sorted cells?
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Detectors can analyze photons without converting them into electrons.
Detectors can analyze photons without converting them into electrons.
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Cells with higher activity levels are generally less fragile.
Cells with higher activity levels are generally less fragile.
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Lasers excite ______ by using specific wavelengths.
Lasers excite ______ by using specific wavelengths.
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What is a major requirement for fluorescent-based sorting?
What is a major requirement for fluorescent-based sorting?
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Mass cytometry can theoretically analyze up to 100 parameters.
Mass cytometry can theoretically analyze up to 100 parameters.
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What does index sorting allow researchers to do with single cells?
What does index sorting allow researchers to do with single cells?
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In mass cytometry, antibodies bind to specific __________ in and at the cells.
In mass cytometry, antibodies bind to specific __________ in and at the cells.
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Match the following techniques with their features:
Match the following techniques with their features:
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What is the primary purpose of compensation in fluorescence applications?
What is the primary purpose of compensation in fluorescence applications?
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Negative controls are used to help discriminate between specific staining and unspecific background.
Negative controls are used to help discriminate between specific staining and unspecific background.
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What types of antibodies should be used as isotype controls?
What types of antibodies should be used as isotype controls?
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The _____ of fluorochromes needs to match expression levels of the antigen in multicolour experiments.
The _____ of fluorochromes needs to match expression levels of the antigen in multicolour experiments.
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Match the following types of negative controls with their descriptions:
Match the following types of negative controls with their descriptions:
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Which filter is used with alexa 488 in the described compensation?
Which filter is used with alexa 488 in the described compensation?
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Compensation is unnecessary in experiments where only one fluorophore is used.
Compensation is unnecessary in experiments where only one fluorophore is used.
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Name one type of background that negative controls can help identify.
Name one type of background that negative controls can help identify.
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In multicolour experiments, the rescue of MCSFR-/- progenitors indicates their response to _____.
In multicolour experiments, the rescue of MCSFR-/- progenitors indicates their response to _____.
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What issue do negative controls help to address in fluorescence applications?
What issue do negative controls help to address in fluorescence applications?
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Study Notes
FACS (Fluorescent Activated Cell Sorting)
- FACS is a technique analyzing physical and chemical properties of single particles (cells)
- It allows for separation of single particles from a heterogeneous population
Flow Cytometry
- Analyzes physical and chemical properties of single particles (cells)
- Particles pass a voltage or light source at high speed
- Allows separation of single particles from a heterogeneous population
Other Separation Forms for Cell Populations
- Cell filtration
- Cell affinity methods
- Fractioning
- Elutration
- MACS (Magnetic Activated Cell Sorting)
- FACS is versatile (uses optics)
- FACS can analyze and sort up to 4 populations simultaneously
- FACS machines are expensive
- Requires a trained user
What to Analyze and Sort for?
- Mammalian cells, yeast, bacteria, plant cells
- Subcellular organelles (Golgi complex, chromosomes, sperm)
- Analysis: size/volume/complexity, cell cycle, RNA/DNA content, protein expression, enzymatic activity, apoptosis, MDR in cancer cells
- Sorting: cell culture, functional assays, transplantation, microarray analysis, single cell-PCR, cloning
Which Parameters are Measured with FACS?
- Blood cells
- Chromosomes
- Algae
- Protozoa
Parameters (more detail)
- Cells are morphologically distinct based on size and granularity.
- Specific size and granularity measurements are provided for cell types
- Basophils, granulocytes, erythrocytes, eosinophils, granulocytes, monocytes, neutrophils, granulocytes
Cellular Parameters: Relative Size and Complexity
-
Forward Scatter (FSC): Measured along the axis of incoming light
- Proportional to cell size/cell surface area (true for perfectly round cells)
-
Side Scatter (SSC): Measured in 90° direction to excitation light
- Proportional to cell complexity or granularity
Cellular Parameters: Example for Light Scattering in Whole Blood
- Graph depicting the relationship between forward scatter and side scatter for blood cells
- Differentiating blood cell types are shown (granulocytes, monocytes, lymphocytes)
Cellular Parameters: Fluorochrome Detection and Quantification
- Fluorochrome molecule absorbs energy from incoming light
- Releases this energy as vibration and dissipated heat
- Emits a photon of a lower wavelength
Cellular Parameters: Fluorochrome Detection and Quantification (cont.)
- Fluorescent signals are proportional to the number of fluorochromes bound to cells.
FACS Machine Components
- Optical system
- Fluidics
- Electronics
Optical System
- Lasers (488nm, 405nm, 635nm, etc.)
- Optical fibers
- Prisms
- Focus lenses
- Flow cell
- Optical fibers
- Filters
- Detectors
Optical System: Lasers
- Excitation of fluorchromes by different lasers
- Various dye excitation by different wavelengths
Optical System: Filters
- Long-pass filters (580 nm LP)
- Short-pass filters (580 nm SP)
- Neutral-density filters (ND1)
- Band-pass filters (660/20 BP)
- Photon distribution to detectors is filter dependent
Optical System: Detectors
- Photodiodes and Photomultiplier Tubes (PMTs)
- Convert photons into electrons (electrical signals)
- Efficiency of conversion dependent on wavelength
- Amplification inside the PMT
Fluidics
- Sheath flow
- Sample flow
- Flow cell
- Interrogation point
- Sample collection chamber
- Waste aspirator
- Collection tube/plate
- Hydrodynamic focusing
Fluidics-Drop Formation and Charging
- Sample reaches cuvette
- Passes through nozzle
- Vibration separates into single droplets
- Droplets pass laser (analysis)
- Droplets are electrically charged
- Droplets pass deflection plates
- Collection in collection tubes or pass to waste
Electronics
- Collecting and display data
Electronics (cont.)
- Pulse width, pulse height, pulse area
- Electron converted into numerical values
- Event intensity depicted as numerical values
Electronics: Graphically Depicting Data
- Histogram
- Dotplot
Electronics: Dot Plots
- 2-dimensional representation for correlation of 2 parameters
- Possible outcomes based on correlation outcome (single positive, double positive, double negative)
Examples Dotplots
- Examples include gates for dead cells, cell doublets, fluorophores, and doubly-labeled cells
Sorting (general principles)
- Stream stability depends on precise adaptation of sheath pressure, frequency, nozzle size, and amplitude
Sorting (general principles)
- Frequency (number of droplets per second) relates to the speed of the sorting process
- Nozzle size and sheath pressure influence cell survival
- Cell type characteristics affect process for successful outcome.
- Specific cell type characteristics relate to corresponding nozzle size and pressure
Sort Setup: How to Design a FACS Experiment
- Selecting cells to analyze and determining appropriate nozzle size
- Cell preparation methods (suspension, cell culture, adherent cells, solid tissue)
- Success of FACS Sort is dependent on the sample condition
- Cells or particles should be adequately separated for the success of FACS experiment
Sort Setup: What to Use Sorted Cells For
- Cell culture
- Functional assays
- Transplantation
- Microarray analysis
- Single-cell PCR
- Cloning
Sort Setup: Analysis Parameters
- Antigen expression, Fluorescent protein expression, Nucleic acid content, Functionality
- Choice of antibodies, fluorescent dyes, or fluorochromes
Compensation
- Correction of spectral overlap between fluorochromes
- Necessary when analyzing samples with multiple markers
Compensation examples
- Spectral overlap of Alexa 488 and PE
Gating and Negative Controls
- Differentiating between specific staining and nonspecific background
- Example with GFP, prominin 1, and PE staining
Negative Controls
- Isotype controls, unstained samples, wildtype animals, control transfections.
Optimal Choice of Fluorochromes (Multicolor Experiment)
- Choosing appropriate fluorochromes to match antigen expression levels
FACS Experiment-Process and Result
FACS issues - Index Sorting
- FSC and SCS are only general cell properties
- Establishing appropriate parameters for permeabilization, labeling
- Requires prior knowledge of cell-specific markers
- Antibody availability and purity
Index sorting
- Allows isolating single cells for retrospective analysis
- High dimensional immune phenotype
- Retrospective analysis via mass spectrometry or single-cell sequencing
Single-Cell Mass Cytometry
- Mass cytometry utilizes antibodies coupled to isotopes of transition elements
- Cells are vaporized and ionized to quantify atomic components
- Up to 100 parameters can be analyzed, combinable with FACS and Phospho-FACS
Image Activated Cell Sorting (IACS)
- Cell type purification through SC transcriptome training
- Uses scRNA-seq data for unbiased cell type identification
- Predicts optimal gating for FACS experiments
Latest Developments in Flow Cytometry
- Intracellular flow cytometry (Phospho-FACS)
- Quantum-Dot technology
- Single-cell mass cytometry
- Image-activated cell sorting (IACS)
Intracellular Flow Cytometry (Phospho-FACS)
- Analyzing intracellular phosphorylation-dependent signaling pathways
- Diagnostic tests, statistics of signaling cascades, and drug screening
- Specific staining protocol using antibodies against cell surface proteins, fixation, steps for permeabilization, and intracellular staining
Quantum-Dot Technology
- Uses nano-crystals in place of fluorochromes
- Advantages: excitable by any laser, 90% of absorbed energy emitted as light, symmetric spectra, low spectral overlap
- Disadvantages: difficulty in production, limited antibody availability
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Description
This quiz covers the fundamental concepts of Fluorescence-Activated Cell Sorting (FACS), including its uses, components, and operational principles. Test your knowledge on parameters measured by FACS and the roles of various system components in fluidics and optics.
