Skip to main content

Cytometry

Functional Assays

Overview of Functional Assays

  • Definition: Functional assays in flow cytometry measure the activity or function of cells, providing insights into their physiological state, signaling pathways, and response to stimuli
  • Purpose:
    • Assess Cell Activity: To measure the production of cytokines, the expression of activation markers, or the activity of signaling pathways
    • Study Cell Function: To investigate the mechanisms of cell function and the effects of various stimuli
    • Diagnose Diseases: To diagnose and monitor diseases based on abnormal cell function
    • Evaluate Therapies: To evaluate the effectiveness of therapeutic interventions
  • Common Types of Functional Assays:
    • Cytokine Assays
    • Chronic Granulomatous Disease (CGD) Assay
    • Calcium Flux Assay
    • Phospho Flow Assay

Cytokine Assays

  • Definition: Cytokine assays measure the production of cytokines by cells, providing information about their activation state and function
  • Types:
    • Intracellular Cytokine Staining (ICS):
      • Technique: Cells are stimulated to produce cytokines, then fixed and permeabilized to allow antibodies to access intracellular cytokines
      • Advantages: Can measure cytokine production by specific cell populations
      • Disadvantages: Requires cell stimulation and permeabilization, which can affect cell viability and antibody binding
    • Cytokine Secretion Assays:
      • Technique: Cells are stimulated to secrete cytokines, which are then captured by antibodies on the cell surface or on beads
      • Advantages: Can measure cytokine secretion without cell permeabilization
      • Disadvantages: Requires specialized antibodies and may not be as sensitive as ICS
  • Considerations:
    • Stimulation Conditions: The choice of stimulus, concentration, and duration can affect cytokine production
    • Blocking Agents: Use blocking agents to prevent cytokine secretion, allowing for accumulation of cytokines within the cell
    • Antibody Selection: Choose antibodies that are specific for the target cytokines and that have been validated for flow cytometry
  • Common cytokines:
    • IL-2
    • IL-4
    • IL-6
    • TNF-alpha
    • IFN-gamma
  • How to optimize:
    • Stimulating the cells effectively with PMA and ionomycin for the appropriate amount of time
    • Using GolgiPlug to retain the signal inside of the cells

Chronic Granulomatous Disease (CGD) Assay

  • Definition: CGD is a genetic disorder that affects the ability of phagocytes (e.g., neutrophils, macrophages) to produce reactive oxygen species (ROS)
  • Assay Principle:
    • The CGD assay measures the production of ROS by phagocytes in response to stimulation
    • Cells are stimulated with phorbol myristate acetate (PMA) or other stimuli, and then incubated with a dye that fluoresces upon oxidation by ROS
    • Flow cytometry is used to measure the fluorescence intensity of the cells, which is proportional to the amount of ROS produced
  • Considerations:
    • Stimulation Conditions: The choice of stimulus, concentration, and duration can affect ROS production
    • Dye Selection: Choose a dye that is sensitive to ROS and that has minimal spectral overlap with other fluorochromes in the panel
    • Controls: Include positive and negative controls to validate the assay
  • Reagents and Supplies:
    • Dihydrorhodamine 123 (DHR): a nonfluorescent cell-permeable dye that can be oxidized by intracellular ROS to form the fluorescent product rhodamine
  • Helpful Hints:
    • Whole blood samples must be tested within a reasonable timeframe
    • Proper amount of DHR and PMA must be used
    • The incubation temperature of the cell suspension must be at 37C

Calcium Flux Assay

  • Definition: The calcium flux assay measures the changes in intracellular calcium concentration in response to stimulation
  • Assay Principle:
    • Cells are loaded with a calcium-sensitive dye, such as Fluo-4 or Indo-1
    • The cells are then stimulated with an agonist that triggers calcium release from intracellular stores or influx from the extracellular environment
    • Flow cytometry is used to monitor the fluorescence intensity of the cells over time, which is proportional to the intracellular calcium concentration
  • Considerations:
    • Dye Loading: Proper dye loading is essential for accurate measurements
    • Stimulation Conditions: The choice of agonist, concentration, and duration can affect calcium flux
    • Instrument Setup: The flow cytometer must be set up to acquire data rapidly over time
  • How to optimize:
    • Use Indo-1 as a ratiometric dye to avoid the need to wash the dye
    • Include a stimulant and ionophore to maximize the signal
    • Maintain the cells at 37C throughout the run

Phospho Flow Assay

  • Definition: The phospho flow assay measures the phosphorylation status of intracellular proteins, providing information about the activation of signaling pathways
  • Assay Principle:
    • Cells are stimulated to activate signaling pathways, then fixed and permeabilized to allow antibodies to access intracellular phosphoproteins
    • The cells are then stained with antibodies that specifically recognize phosphorylated forms of the target proteins
    • Flow cytometry is used to measure the fluorescence intensity of the cells, which is proportional to the level of phosphorylation
  • Considerations:
    • Stimulation Conditions: The choice of stimulus, concentration, and duration can affect protein phosphorylation
    • Fixation and Permeabilization: Proper fixation and permeabilization are essential for preserving phosphoproteins
    • Antibody Selection: Choose antibodies that are specific for the phosphorylated forms of the target proteins and that have been validated for flow cytometry
    • Add phosphatase inhibitors: Ensure that the cell medium or staining buffer is supplemented with phosphatase inhibitors to minimize unwanted dephosphorylation

General Considerations for Functional Assays

  • Cell Viability:
    • Many functional assays require live cells, so it is essential to maintain high cell viability throughout the experiment
    • Use appropriate cell culture techniques and handling procedures to minimize cell stress
    • Include a viability dye to exclude dead cells from analysis
  • Controls:
    • Include appropriate controls to validate the assay and account for background noise
    • Unstimulated controls, positive controls, and isotype controls are all important for functional assays
    • For example, a positive control can be produced with the use of phorbol myristate acetate (PMA) and ionomycin
  • Compensation:
    • Spectral overlap is a common problem in flow cytometry, and it is essential to use proper compensation techniques to correct for spillover
    • Use single-stain controls to accurately calculate compensation values

Troubleshooting Functional Assay Issues

  • No Response:
    • Possible Causes:
      • Ineffective stimulus
      • Non-responsive cells
      • Incorrect assay protocol
    • Troubleshooting Steps:
      • Optimize stimulation conditions
      • Use positive controls to verify cell responsiveness
      • Review assay protocol
  • High Background:
    • Possible Causes:
      • Non-specific antibody binding
      • Autofluorescence
      • Contamination
    • Troubleshooting Steps:
      • Use blocking reagents
      • Reduce autofluorescence
      • Clean samples
  • Variable Results:
    • Possible Causes:
      • Inconsistent technique
      • Instrument instability
      • Sample heterogeneity
    • Troubleshooting Steps:
      • Standardize assay protocols
      • Calibrate instrument
      • Increase sample size

Key Terms

  • Functional Assay: An assay that measures the activity or function of cells
  • Cytokine: A signaling molecule that mediates communication between cells
  • Reactive Oxygen Species (ROS): Molecules that are produced during cellular metabolism and that can damage cells
  • Calcium Flux: Changes in intracellular calcium concentration
  • Phosphorylation: The addition of a phosphate group to a protein
  • Stimulation: The process of activating cells with a specific stimulus