Skip to main content

Cytometry

Prep and Stain

Overview of Sample Preparation and Staining

  • Definition: Sample preparation involves the steps taken to process a sample before staining, including disaggregation, lysing, filtering, and fixation. Staining involves labeling cells with fluorescent antibodies or dyes to identify specific cell populations or cellular components
  • Importance:
    • Accurate Results: Proper sample preparation and staining are essential for accurate and reliable results
    • Optimal Signal: Proper staining techniques can maximize signal intensity and minimize background noise
    • Cell Preservation: Proper fixation can preserve cell morphology and prevent degradation
  • Key Steps:
    • Disaggregation
    • Lysing Agents
    • Aggregates
    • Filtering
    • Fixation
    • Permeabilization
    • Staining

Disaggregation

  • Definition: The process of breaking down solid tissues or cell clumps into a single-cell suspension
  • Methods:
    • Mechanical Disaggregation:
      • Technique: Using scalpels, needles, grinders, or other instruments to manually break down the tissue
      • Advantages: Simple and inexpensive
      • Disadvantages: Can be time-consuming and may damage cells
    • Enzymatic Digestion:
      • Technique: Using enzymes such as collagenase, trypsin, or dispase to digest the extracellular matrix and release cells
      • Advantages: More efficient and less damaging to cells than mechanical disaggregation
      • Disadvantages: Can be expensive and may require optimization for different tissue types
    • Combination Methods:
      • Technique: Using a combination of mechanical and enzymatic methods to disaggregate the tissue
      • Advantages: Can be more effective than either method alone
      • Disadvantages: Can be more complex and time-consuming
  • Considerations:
    • Tissue Type: The choice of disaggregation method depends on the type of tissue being processed
    • Enzyme Concentration: The concentration of enzyme used should be optimized to minimize cell damage
    • Incubation Time: The incubation time should be optimized to ensure complete disaggregation without over-digestion

Lysing Agents

  • Definition: Chemicals used to selectively lyse (break open) certain cell types, such as red blood cells (RBCs)
  • Purpose:
    • Remove Unwanted Cells: To remove RBCs from blood samples, allowing for better visualization of other cell populations
    • Isolate Intracellular Components: To release intracellular proteins or nucleic acids for analysis
  • Common Lysing Agents:
    • Ammonium Chloride (NH4Cl): A hypotonic solution that causes RBCs to swell and burst
    • Saponin: A detergent that disrupts cell membranes
    • Commercial Lysing Buffers: Pre-formulated buffers that contain a mixture of lysing agents
  • Considerations:
    • Cell Sensitivity: Different cell types have different sensitivities to lysing agents
    • Incubation Time: The incubation time should be optimized to ensure complete lysis of unwanted cells without damaging target cells
    • Buffer Composition: The composition of the lysing buffer can affect cell viability and antibody binding

Aggregates

  • Definition: Clumps of cells that can interfere with flow cytometry analysis
  • Causes:
    • Improper Sample Preparation: Inadequate disaggregation or handling
    • Cell Death: Dead cells can clump together and form aggregates
    • Antibody Aggregation: Antibodies can aggregate and bind non-specifically to cells
  • Methods to Minimize Aggregates:
    • Proper Disaggregation: Ensure complete disaggregation of tissues or cell clumps
    • DNAse treatment: To remove any extracellular DNA that may cause cells to stick together
    • Gentle Handling: Avoid vigorous shaking or vortexing of samples
    • Filtering: Filter samples through a cell strainer to remove aggregates
    • DNAse: Treat samples with DNAse to digest DNA released from dead cells, which can cause aggregation
  • Strategies to Identify and Exclude Aggregates:
    • Gating on Forward Scatter (FSC) Parameters: Use FSC-A (area) vs. FSC-H (height) or FSC-W (width) to identify and exclude aggregates
    • Visual Inspection: Examine samples under a microscope to identify aggregates

Filtering

  • Definition: The process of passing a sample through a filter to remove large particles, debris, and aggregates
  • Purpose:
    • Remove Debris: To improve data quality by reducing background noise and non-specific binding
    • Prevent Clogging: To prevent clogging of the flow cytometer nozzle
    • Reduce Aggregates: To remove cell aggregates that can interfere with analysis
  • Filter Size:
    • Typically 40 μm to 70 μm, depending on the size of the cells being analyzed
  • Filter Material:
    • Nylon, polyester, or other materials that are compatible with flow cytometry
  • Considerations:
    • Cell Loss: Filtration can result in some loss of cells, especially if the filter is too small
    • Filter Clogging: Filters can clog if the sample contains a lot of debris
  • Filtering by gravity:
    • Cell suspensions with a volume greater than 1mL
  • Filtering using syringe:
    • Cell suspensions with a volume less than 1mL

Fixation

  • Definition: The process of treating cells with chemicals to preserve their structure and prevent degradation
  • Purpose:
    • Preserve Morphology: To maintain the shape and size of cells
    • Prevent Degradation: To prevent the breakdown of cellular components
    • Inactivate Enzymes: To stop enzymatic activity that can alter cell phenotype
    • Allow for Storage: To allow samples to be stored for later analysis
  • Common Fixatives:
    • Formaldehyde: A commonly used fixative that cross-links proteins
    • Paraformaldehyde (PFA): A polymer of formaldehyde that is less toxic and less likely to cause cell shrinkage
    • Glutaraldehyde: A stronger fixative than formaldehyde that is often used for electron microscopy
    • Methanol: An alcohol-based fixative that precipitates proteins
  • Considerations:
    • Fixation Time: The fixation time should be optimized to ensure adequate preservation without over-fixation
    • Fixative Concentration: The concentration of fixative used can affect cell morphology and antibody binding
    • Washing Steps: Washing steps are necessary to remove excess fixative and prevent interference with staining
  • Common Fixation Methods:
    • Intracellular staining of cytokines:
      • The samples can be fixed with 2% formaldehyde for 10 minutes
      • This will immobilize the cells without compromising the recognition of the intracellular molecule
    • Transcription factor staining:
      • The samples are fixed with formaldehyde to immobilize the cells
      • The cells are permeabilized with methanol to allow the antibody access to the nucleus

Permeabilization

  • Definition: The process of creating pores in the cell membrane to allow antibodies to access intracellular targets
  • Purpose:
    • Intracellular Staining: To stain proteins, nucleic acids, or other molecules located inside the cell
  • Common Permeabilization Agents:
    • Saponin: A detergent that disrupts cell membranes
    • Triton X-100: A non-ionic detergent that creates pores in the cell membrane
    • Methanol: An alcohol-based fixative that also permeabilizes cells
  • Considerations:
    • Permeabilization Time: The permeabilization time should be optimized to ensure adequate antibody access without damaging the cell
    • Permeabilization Agent Concentration: The concentration of permeabilization agent used can affect cell morphology and antibody binding
    • Washing Steps: Washing steps are necessary to remove excess permeabilization agent and prevent interference with staining
  • Sequential steps for fixation and permeabilization:
    • Cell-surface staining
    • Fixation
    • Wash
    • Permeabilization
    • Intracellular staining
    • Wash

Staining

  • Definition: The process of labeling cells with fluorescent antibodies or dyes to identify specific cell populations or cellular components
  • Steps:
    1. Blocking: Block non-specific binding sites to reduce background noise
    2. Antibody Incubation: Incubate cells with fluorescent antibodies that bind to specific cell surface or intracellular markers
    3. Washing: Wash away unbound antibodies
    4. Counterstaining: Use a counterstain (e.g., DAPI) to label all cells in the sample
  • Considerations:
    • Antibody Selection: Choose antibodies that are specific for the target antigen and that have been validated for flow cytometry
    • Antibody Titration: Titrate antibodies to determine the optimal concentration for staining
    • Fluorophore Selection: Choose fluorophores that have minimal spectral overlap and that are compatible with the flow cytometer’s lasers and detectors
    • Staining Time and Temperature: Optimize staining time and temperature to maximize signal intensity and minimize background noise
    • Controls: Use appropriate controls to validate the staining protocol and to correct for background fluorescence
  • Helpful Tip:
    • When performing surface staining, block the Fc receptors with Fc block to prevent nonspecific binding of the antibodies
    • Incubate the antibody cocktail on ice for 30 minutes to prevent endocytosis and capping of the antibody

Troubleshooting Sample Preparation and Staining Issues

  • Low Event Count:
    • Possible Causes:
      • Cell loss during preparation
      • Clogged nozzle
      • Instrument malfunction
    • Troubleshooting Steps:
      • Optimize sample preparation protocols
      • Filter samples to remove debris
      • Inspect instrument for malfunctions
  • High Background Noise:
    • Possible Causes:
      • Non-specific antibody binding
      • Autofluorescence
      • Contamination
    • Troubleshooting Steps:
      • Use blocking reagents
      • Reduce autofluorescence
      • Clean samples
  • Poor Resolution:
    • Possible Causes:
      • Cell aggregates
      • Incorrect staining
      • Instrument malfunction
    • Troubleshooting Steps:
      • Reduce cell aggregates
      • Optimize staining protocols
      • Inspect instrument for malfunctions
  • 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

Key Terms

  • Disaggregation: Breaking down solid tissues or cell clumps into a single-cell suspension
  • Lysing Agents: Chemicals used to selectively lyse certain cell types
  • Aggregates: Clumps of cells that can interfere with flow cytometry analysis
  • Filtering: Passing a sample through a filter to remove large particles, debris, and aggregates
  • Fixation: Treating cells with chemicals to preserve their structure and prevent degradation
  • Permeabilization: Creating pores in the cell membrane to allow antibodies to access intracellular targets
  • Staining: Labeling cells with fluorescent antibodies or dyes to identify specific cell populations or cellular components