ISAC Analytical Methods and Techniques 2 — Questions and Answers
Question 1: Which compensation method corrects for spectral overlap between fluorochrome channels in flow cytometry?
- Voltage adjustment
- Single-color controls with compensation matrix (Correct answer)
- Threshold gating
- Doublet discrimination
Correct answer: Single-color controls with compensation matrix
Single-color controls are used to build a compensation matrix that mathematically removes signal spillover between detector channels.
Question 2: In spectral flow cytometry, what is the primary advantage over conventional flow cytometry?
- Faster sample acquisition speed
- Full spectral signature capture enabling better unmixing of fluorochromes (Correct answer)
- Lower instrument cost
- Reduced need for antibody titration
Correct answer: Full spectral signature capture enabling better unmixing of fluorochromes
Spectral flow cytometry captures the full emission spectrum of each fluorochrome, allowing autofluorescence separation and better resolution of spectrally similar dyes.
Question 3: What does the term 'staining index' measure in flow cytometry panel design?
- The ratio of positive to negative cell populations
- The separation between positive and negative populations relative to background noise (Correct answer)
- The concentration of antibody used per test
- The total number of events collected
Correct answer: The separation between positive and negative populations relative to background noise
Staining index quantifies how well a fluorochrome separates positive from negative populations by comparing the difference in median fluorescence to the spread of the negative population.
Question 4: Which light source is most commonly used in modern cell sorters for high-speed applications?
- Mercury arc lamp
- Tungsten-halogen lamp
- Solid-state diode laser (Correct answer)
- Xenon flash lamp
Correct answer: Solid-state diode laser
Solid-state diode lasers provide stable, coherent, monochromatic illumination and are standard in high-speed cell sorters due to their reliability and power consistency.
Question 5: When setting up a cell viability assay using a live/dead discriminator dye, why must dead cells be excluded?
- Dead cells absorb too much laser energy and damage the instrument
- Dead cells bind antibodies non-specifically, causing false positives (Correct answer)
- Dead cells are too large to pass through the flow cell
- Dead cells lack surface antigens entirely
Correct answer: Dead cells bind antibodies non-specifically, causing false positives
Dead cells have compromised membranes that allow non-specific antibody binding, leading to artifactual false-positive staining and misidentification of cell populations.
Question 6: In mass cytometry (CyTOF), what replaces fluorescent labels?
- Quantum dots
- Heavy metal isotope tags (Correct answer)
- Enzyme substrates
- Radioactive phosphorus
Correct answer: Heavy metal isotope tags
Mass cytometry uses antibodies conjugated to heavy metal isotopes detected by time-of-flight mass spectrometry, eliminating spectral overlap limitations.
Question 7: Which parameter is used to trigger event acquisition in a flow cytometer when analyzing small particles?
- Side scatter height (SSC-H)
- Forward scatter height (FSC-H)
- Fluorescence channel threshold (Correct answer)
- Pulse width
Correct answer: Fluorescence channel threshold
For small particles like platelets or microvesicles that may fall below FSC threshold sensitivity, fluorescence triggering provides better event detection specificity.
ISAC Analytical Methods and Techniques 2
Which compensation method corrects for spectral overlap between fluorochrome channels in flow cytometry?