Skip to main content

Cytometry

Sample

Sample: The Foundation of Flow Cytometry Experiments

The sample is the foundation of any flow cytometry experiment. The quality and characteristics of your sample directly impact the accuracy, reliability, and ultimately, the validity of your results

Before even thinking about fluorophores or gating strategies, we MUST carefully consider:

  • Where our sample comes from (Source)
  • How to keep it in optimal condition (Integrity)
  • How to prepare it for analysis (Preparation & Staining)
  • Whether we need to isolate specific cells (Enrichment)

The Four Pillars of Sample Design

  1. Sample Source:
    • What it is: The origin of your sample (blood, tissue, cultured cells, etc.)
    • Why it matters: Dictates the appropriate preparation methods, antibody selection, and data interpretation
    • Considerations: Cell types present, potential contaminants, and ethical considerations
  2. Sample Integrity:
    • What it is: The quality and condition of the sample at the time of analysis
    • Why it matters: Ensures accurate, reliable, and reproducible results
    • Key factors: Collection technique, handling procedures, storage conditions, and cell viability
  3. Sample Preparation & Staining:
    • What it is: The steps taken to process a sample before analysis, including disaggregation, lysing, filtering, fixation, permeabilization, and antibody staining
    • Why it matters: Removes unwanted cells and debris, preserves cell morphology, allows access to intracellular targets, and labels cells with fluorescent markers
    • Considerations: Cell type, target antigens, antibody selection, fluorophore selection, and control samples
  4. Cell Enrichment:
    • What it is: The process of increasing the proportion of specific cell types in a sample
    • Why it matters: Isolates rare cells, reduces complexity, and improves signal-to-noise ratio
    • Techniques: Cell sorting (FACS), density gradient isolation, and magnetic bead separation (MACS)

How They Work Together

These four pillars aren’t independent; they’re interconnected and influence each other:

  1. The Sample Source determines the necessary Preparation steps (e.g., solid tissue requires disaggregation, blood requires RBC lysis)
  2. Proper Collection and Handling are crucial for maintaining Sample Integrity throughout the experiment
  3. Enrichment may be necessary if your target cells are rare within the initial Sample Source
  4. The choice of Fixation and Permeabilization methods depends on the target antigens and the antibodies used for Staining

Why These Considerations are Critical

  • Accurate Data: By carefully considering these four aspects, you’re setting the stage for obtaining accurate and meaningful data
  • Reliable Results: Consistent and well-controlled sample handling leads to reproducible results
  • Optimized Experiments: Thoughtful sample design allows you to tailor your experiment to your specific research question, maximizing the information you can extract
  • Valid Conclusions: Ultimately, high-quality sample design enables you to draw valid conclusions about the biological system you’re studying

Troubleshooting Sample Issues

  • Low Event Count: Check sample source, preparation, viability, and instrument settings
  • High Background Noise: Optimize preparation to eliminate non-specific binding
  • Poor Resolution: Adjust your preparation/staining techniques
  • Unexpected Results: Question everything! Re-examine your sample source, preparation, and staining protocols

Key Takeaways

  • A well-designed flow cytometry experiment begins with careful consideration of the sample
  • Sample source, integrity, preparation & staining, and cell enrichment are critical factors that influence the quality and reliability of the data
  • By paying attention to these four pillars, you can optimize your experiments and ensure that you’re drawing valid conclusions