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Cytometry

Mass Cytometry

Overview of Mass Cytometry (CyTOF)

  • Definition: Mass cytometry, also known as cytometry by time-of-flight (CyTOF), is a technique that uses antibodies conjugated to heavy metal isotopes to quantify multiple cellular markers simultaneously
  • Principle:
    • Cells are labeled with antibodies conjugated to unique heavy metal isotopes
    • The cells are introduced into an inductively coupled plasma mass spectrometer (ICP-MS)
    • The ICP-MS atomizes and ionizes the cells, and then measures the mass-to-charge ratio of the metal isotopes
    • The abundance of each metal isotope is proportional to the amount of antibody bound to the cell, which in turn is proportional to the expression level of the target marker
  • Advantages Over Traditional Flow Cytometry:
    • High Dimensionality: Can measure 40+ markers simultaneously, compared to the limited number of fluorochromes that can be used in traditional flow cytometry
    • Minimal Spectral Overlap: Uses mass-to-charge ratio instead of fluorescence, eliminating the problem of spectral overlap
    • High Sensitivity: Can detect low levels of protein expression
  • Disadvantages:
    • Lower Throughput: Slower than traditional flow cytometry
    • Cell Destruction: The cells are destroyed during the ionization process, preventing cell sorting
    • Specialized Equipment: Requires a specialized mass cytometer
    • High Cost: More expensive than traditional flow cytometry
  • Applications:
    • Immunology
    • Cancer Research
    • Stem Cell Research
    • Drug Discovery

Key Components of a Mass Cytometer

  • Fluidics System:
    • Transports the cells from the sample tube to the nebulizer
    • Maintains a stable and consistent flow rate
  • Nebulizer:
    • Converts the liquid sample into a fine aerosol
  • Inductively Coupled Plasma (ICP):
    • A high-temperature plasma that atomizes and ionizes the sample
    • The ICP is generated by passing argon gas through a radiofrequency field
  • Mass Analyzer:
    • Separates the ions based on their mass-to-charge ratio
    • The mass analyzer in a CyTOF instrument is typically a time-of-flight (TOF) mass spectrometer
  • Detector:
    • Detects the ions and measures their abundance
    • The detector in a CyTOF instrument is typically an electron multiplier
  • Software System:
    • Controls the instrument
    • Acquires and processes the data
    • Provides data visualization and analysis tools

Antibodies and Metal Conjugation

  • Antibodies:
    • Use high-quality antibodies that are specific for the target markers
    • Validate antibodies for use in mass cytometry
  • Metal Conjugation:
    • Antibodies are conjugated to unique heavy metal isotopes using specialized conjugation kits
    • The choice of metal isotope depends on its abundance, purity, and the availability of conjugation chemistry
  • Examples of Metal Isotopes:
    • Lanthanides: 139La, 141Pr, 142Nd, 143Nd, 144Nd, 145Nd, 146Nd, 147Sm, 148Sm, 149Sm, 150Nd, 151Eu, 152Sm, 153Eu, 154Sm, 155Gd, 156Gd, 157Gd, 158Gd, 159Tb, 160Gd, 161Dy, 162Dy, 163Dy, 164Dy, 165Ho, 166Er, 167Ho, 168Er, 169Tm, 170Er, 171Yb, 172Yb, 173Yb, 174Yb, 175Lu, 176Yb
  • Considerations:
    • Metal Purity: Use high-purity metal isotopes to minimize background noise
    • Conjugation Efficiency: Optimize the conjugation reaction to maximize the number of metal atoms per antibody molecule
    • Antibody Titration: Titrate the metal-conjugated antibodies to determine the optimal concentration for staining
  • Barcoding and debarcoding:
    • Use of a metal conjugated to a DNA linker to label the sample
    • This allows for multiple samples to be combined and to be analyzed at the same time, saving time and resources
    • This needs to be removed from the data prior to analysis

Sample Preparation for Mass Cytometry

  • Cell Staining:
    • Label cells with metal-conjugated antibodies
    • Use appropriate staining protocols to maximize signal intensity and minimize background noise
  • Fixation and Permeabilization:
    • Fix and permeabilize the cells to preserve cell structure and allow antibody access to intracellular targets
    • Use appropriate fixation and permeabilization methods for the target antigens
  • DNA Intercalation:
    • Intercalate a metal-labeled DNA intercalator (e.g., iridium) into the DNA to allow for cell identification and doublet discrimination
  • Washing:
    • Wash the cells thoroughly to remove unbound antibodies and other contaminants
  • Filtration:
    • Filter the sample through a 35 um filter
  • Cell Concentration:
    • Ensure that the cell concentration is within the optimal range for the mass cytometer

Data Analysis for Mass Cytometry

  • Normalization:
    • Normalize the data to account for variations in instrument performance over time
    • Use normalization beads to correct for signal drift
  • Deconvolution:
    • Deconvolve the data to remove signal from metal isotopes that have overlapping mass-to-charge ratios
  • Gating:
    • Use a hierarchical gating strategy to identify specific cell populations based on their marker expression
    • Use appropriate controls to define gating boundaries
  • Visualization:
    • Use visualization tools to explore the high-dimensional data
    • Common visualization methods include t-distributed stochastic neighbor embedding (t-SNE), uniform manifold approximation and projection (UMAP), and viSNE
  • Clustering:
    • Use clustering algorithms to identify groups of cells with similar marker expression profiles
    • Common clustering algorithms include k-means clustering and hierarchical clustering

Applications of Mass Cytometry

  • Immunology:
    • Deep phenotyping of immune cell populations
    • Studying immune responses to infection and vaccination
    • Investigating the mechanisms of autoimmune diseases
  • Cancer Research:
    • Identifying and characterizing cancer stem cells
    • Studying the tumor microenvironment
    • Developing new cancer therapies
  • Stem Cell Research:
    • Characterizing stem cell differentiation and self-renewal
    • Identifying novel stem cell markers
    • Developing new stem cell therapies
  • Drug Discovery:
    • Screening for drugs that affect cellular signaling pathways
    • Identifying drug targets

Troubleshooting Mass Cytometry Assays

  • Low Event Rate:
    • Possible Causes:
      • Clogged nebulizer
      • Low cell concentration
      • Instrument malfunction
    • Troubleshooting Steps:
      • Clean nebulizer
      • Increase cell concentration
      • Inspect instrument for malfunctions
  • High Background Noise:
    • Possible Causes:
      • Contaminated reagents
      • Poor antibody staining
      • Instrument settings
    • Troubleshooting Steps:
      • Use clean reagents
      • Optimize antibody staining
      • Adjust instrument settings
  • Unexpected Results:
    • Possible Causes:
      • Incorrect antibody selection
      • Improper sample preparation
      • Instrument malfunction
    • Troubleshooting Steps:
      • Verify antibody specificity
      • Review sample preparation protocols
      • Inspect instrument for malfunctions
  • Data Normalization Issues:
    • Possible Causes:
      • Poor bead distribution
      • Insufficient events acquired
    • Troubleshooting Steps:
      • Improve bead mixing in samples
      • Acquire adequate number of events