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Cytometry

Fluorochrome Selection

Overview of Fluorochrome Selection

  • Definition: Fluorochrome selection involves choosing the appropriate fluorescent dyes to label cells for flow cytometry analysis. The choice of fluorochromes depends on several factors, including the expression level of the target antigen, the spectral properties of the fluorochromes, and the configuration of the flow cytometer
  • Importance:
    • Optimal Signal: Proper fluorochrome selection can maximize signal intensity and minimize background noise, allowing for clear detection and quantification of target cells
    • Spectral Separation: Fluorochromes should have minimal spectral overlap to allow for accurate compensation and resolution of different cell populations
    • Panel Design: Fluorochrome selection is an integral part of panel design, ensuring that the chosen fluorochromes are compatible with each other and with the flow cytometer’s lasers and detectors
  • Key Considerations:
    • Antigen Density
    • Protein Coexpression
    • Optimal Combination
    • Photostability
    • F/P Ratio
    • Quenching
    • Signal to Noise Ratio

Antigen Density

  • Definition: The amount of target antigen present on the cell surface or within the cell
  • Impact on Fluorochrome Selection:
    • High Antigen Density: Use dimmer fluorochromes for highly expressed antigens to avoid oversaturation of the signal
    • Low Antigen Density: Use brighter fluorochromes for weakly expressed antigens to maximize signal intensity
  • Commonly Accepted Practice:
    • Match fluorochrome brightness with relative target expression (bright binds to low density and dim binds to high density)

Protein Coexpression

  • Definition: The simultaneous expression of two or more proteins on the same cell
  • Impact on Fluorochrome Selection:
    • Coexpressed Proteins: Choose fluorochromes that have minimal spectral overlap to allow for accurate discrimination of cells expressing both proteins
    • Mutually Exclusive Proteins: Choose fluorochromes that have good spectral separation to easily distinguish between cells expressing one protein or the other
  • Considerations:
    • Compensation: Spectral overlap can be corrected using compensation, but excessive overlap can reduce resolution
    • Spread: Minimize the spread of fluorescence into other channels by choosing fluorochromes with minimal overlap
  • Protein co-expression relationships:
    • If two or more proteins of interest are known to have a direct co-relationship, be mindful of fluorophore selection to optimize the detection of lower or higher expressed proteins

Optimal Combination

  • Definition: Choosing a combination of fluorochromes that work well together in a flow cytometry panel
  • Considerations:
    • Laser Lines: Choose fluorochromes that are efficiently excited by the lasers available on the flow cytometer
    • Detector Sensitivity: Consider the sensitivity of the detectors for each fluorochrome
    • Spillover: Minimize the spillover of fluorescence from one fluorochrome into another
    • Brightness Hierarchy: Arrange fluorochromes in a brightness hierarchy, with the brightest fluorochromes used for weakly expressed antigens and the dimmest fluorochromes used for highly expressed antigens
  • Panel Design Software:
    • Use panel design software to help select an optimal combination of fluorochromes
    • These programs can predict spectral overlap and suggest fluorochrome combinations that minimize spillover

Photostability

  • Definition: The ability of a fluorochrome to resist fading or bleaching upon exposure to light
  • Impact on Fluorochrome Selection:
    • Photostable Fluorochromes: Choose photostable fluorochromes for experiments that require prolonged exposure to light, such as cell sorting or imaging flow cytometry
    • Photosensitive Fluorochromes: Avoid using photosensitive fluorochromes for experiments that require prolonged exposure to light
  • Common Practice:
    • Minimize exposure to light. When cells are not being analyzed by the instrument, it is important to keep the sample in the dark
    • When the instrument is not being used, be sure to turn off the lasers

F/P Ratio (Fluorophore to Protein Ratio)

  • Definition: The number of fluorochrome molecules attached to each antibody molecule
  • Impact on Signal Intensity:
    • High F/P Ratio: Higher F/P ratio can increase signal intensity but may also increase non-specific binding
    • Low F/P Ratio: Lower F/P ratio can reduce signal intensity but may also decrease non-specific binding
  • Considerations:
    • Antibody Quality: Use high-quality antibodies with a consistent F/P ratio
    • Titration: Titrate antibodies to determine the optimal concentration for staining

Quenching

  • Definition: The reduction in fluorescence intensity due to interactions between fluorochrome molecules or with other molecules in the environment
  • Types:
    • Self-Quenching: Occurs when fluorochrome molecules are too close together, reducing their fluorescence efficiency
    • Environmental Quenching: Occurs when the fluorochrome interacts with molecules in the environment, such as oxygen or water
  • Impact on Fluorochrome Selection:
    • Avoid High Concentrations: Avoid using high concentrations of fluorochromes to minimize self-quenching
    • Use Antioxidants: Use antioxidants to reduce environmental quenching
  • How to avoid quenching:
    • Be sure to protect the sample from light

Signal-to-Noise Ratio (SNR)

  • Definition: The ratio of the signal intensity to the background noise level
  • Impact on Fluorochrome Selection:
    • High SNR: Choose fluorochromes that provide a high SNR to allow for clear detection of target cells
    • Low SNR: Avoid using fluorochromes with low SNR, as they may make it difficult to distinguish target cells from background noise
  • Strategies to Improve SNR:
    • Use brighter fluorochromes
    • Optimize staining protocols
    • Reduce autofluorescence
    • Use appropriate controls to subtract background noise

Spectral Overlap and Compensation

  • Spectral Overlap: Occurs when the emission spectra of two or more fluorochromes overlap, making it difficult to distinguish between them
  • Compensation: A mathematical process used to correct for spectral overlap by subtracting the contribution of one fluorochrome from the signal of another
  • Factors Affecting Compensation:
    • Fluorochrome Selection: Choose fluorochromes with minimal spectral overlap to reduce the need for compensation
    • Spillover Matrices: Properly prepare single-stain controls
  • Compensation Controls:
    • Use single-stain controls to accurately calculate compensation values
    • Consider using automatic compensation algorithms to simplify the process

Troubleshooting Fluorochrome Issues

  • Weak Signal:
    • Possible Causes:
      • Low fluorochrome concentration
      • Incorrect excitation wavelength
      • Photobleaching
    • Troubleshooting Steps:
      • Increase fluorochrome concentration
      • Verify excitation wavelength
      • Protect from light
  • High Background Noise:
    • Possible Causes:
      • Non-specific binding
      • Autofluorescence
      • Contamination
    • Troubleshooting Steps:
      • Use blocking reagents
      • Reduce autofluorescence
      • Clean samples
  • Inaccurate Compensation:
    • Possible Causes:
      • Incorrect compensation values
      • Poor single-stain controls
      • Instrument malfunction
    • Troubleshooting Steps:
      • Verify compensation values
      • Prepare high-quality single-stain controls
      • Inspect instrument for malfunctions

Key Terms

  • Fluorophore: A molecule that emits fluorescence
  • Fluorochrome: A fluorescent dye that emits light when excited by a specific wavelength of light
  • Antigen Density: The amount of target antigen present on the cell surface or within the cell
  • Protein Coexpression: The simultaneous expression of two or more proteins on the same cell
  • Photostability: The ability of a fluorochrome to resist fading or bleaching upon exposure to light
  • F/P Ratio: The number of fluorochrome molecules attached to each antibody molecule
  • Quenching: The reduction in fluorescence intensity due to interactions between fluorochrome molecules or with other molecules in the environment
  • Signal-to-Noise Ratio (SNR): The ratio of the signal intensity to the background noise level
  • Spectral Overlap: Occurs when the emission spectra of two or more fluorochromes overlap
  • Compensation: A mathematical process used to correct for spectral overlap