Flow Cytometry Principles

Flow Cytometry Principles

• Flow cytometry is a technique analyzing cell physical and chemical characteristics as cells pass through a laser beam.
• It has applications in immunophenotyping, cell cycle analysis, and apoptosis detection.
• Fluidics System: Transports cells/particles in a stream, achieving hydrodynamic focusing to align cells for precise measurement.
  • Sheath fluid focuses the sample.
  • Sample injection port introduces the sample.
• Optical System: Excitation by lasers and detection of emitted signals.
  • Laser source excites fluorochromes.
  • Lenses and filters direct and separate light signals.
• Electronics System: Converts optical signals into electronic data.
  • Photodetectors capture light.
  • Signals are amplified, processed, and analyzed by computer.
• Fluorescent Markers: Fluorochromes bind to cellular components (like DNA, proteins) emitting light at specific wavelengths when excited by a laser.
  • Examples: FITC (green), PE (orange-red), APC (far-red).
• Data Analysis: Data visualized in histograms (one parameter, e.g., fluorescence intensity) and dot plots (two parameters, e.g., size vs granularity).
  • Gating identifies subpopulations based on parameters.

Flow Cytometry Applications

• Immunophenotyping: Identifies cell types using specific surface markers.
  • Common in leukemia and lymphoma classification, and monitoring immune cell subsets (like CD4/CD8 ratios in HIV).
• Cell Cycle Analysis: Measures DNA content using stains like Propidium Iodide (PI).
  • Identifies G0/G1, S, and G2/M phases.
  • Applications in cancer research (proliferation studies).
  • Protocol: Fix cells in 75% ethanol, stain with PI and RNase, then analyze fluorescence intensity.
• Apoptosis Detection: Detects markers of programmed cell death.
  • Techniques: Annexin V/PI staining (differentiates live, apoptotic, and necrotic cells), caspase activation assays.
• Stem Cell Research: Monitors CD34+ hematopoietic stem cells for bone marrow transplantation and stem cell therapy.
• Infectious Disease Monitoring: In HIV progression, tracks CD4+ T-cell counts to guide antiretroviral therapy initiation.
• Protocol for Hematopoietic Stem Cell Monitoring: This details a specific procedure.
  • Collect 300μl of EDTA whole blood.
  • Add 15μl of CD34-PE-Ab/CD45-FITC Ab.
  • Incubate for 45 min in the dark.
  • Add 4ml RBC lysis buffer and incubate another 10 min in the dark.
  • Spin at 300g for 10 min to remove lysate, add washing buffer.
  • Spin again at 300g for 10 min, and remove the lysate and add 1ml washing buffer.
  • Perform FACS analysis.

Flow Cytometry Advantages and Limitations

• Advantages: Rapid analysis of thousands of cells per second, multicolor capabilities for multiple markers, high sensitivity and specificity.
• Limitations: Expensive equipment and reagents, skilled technicians required for operation and data interpretation, sample preparation can introduce artifacts.

Emerging Trends in Flow Cytometry

• Spectral Flow Cytometry: Captures the entire fluorescence spectrum, enhancing resolution.
• Mass Cytometry: Combines flow cytometry with mass spectrometry for high-dimensional analysis.
• AI and Machine Learning: Automates data analysis and improves diagnostic accuracy.

Explore the fundamental principles of flow cytometry, a powerful technique used to analyze the physical and chemical characteristics of cells. This quiz covers the fluidics, optical, and electronics systems involved, as well as the role of fluorescent markers in cell analysis. Test your knowledge and understand how these systems work together.