SCYM Data Acquisition & Analysis 5 — Questions and Answers
Question 1: In mass cytometry (CyTOF), heavy metal isotopes replace fluorochromes. Which statement best describes why spillover is minimized in CyTOF?
- Metal isotopes do not produce light
- Atomic mass channels are nearly discrete with minimal spectral overlap, unlike broad fluorochrome emission spectra (Correct answer)
- CyTOF uses fewer detectors
- Metal isotopes are quenched before detection
Correct answer: Atomic mass channels are nearly discrete with minimal spectral overlap, unlike broad fluorochrome emission spectra
Mass-to-charge detection separates isotopes by atomic mass, which have much narrower 'overlap' than optical fluorochrome emission spectra, allowing 40+ parameters with minimal crosstalk.
Question 2: After acquiring data, a researcher finds the negative population in the FITC channel is shifted higher than expected. The MOST likely cause is:
- Too little compensation applied
- Autofluorescence from the cells contributing to the FITC channel signal (Correct answer)
- Insufficient laser power
- Incorrect FSC threshold
Correct answer: Autofluorescence from the cells contributing to the FITC channel signal
Cellular autofluorescence is most prominent in the FITC (green) channel and raises the apparent fluorescence of unstained cells, mimicking dim positive staining.
Question 3: Which parameter is most commonly used as the trigger (threshold) channel in standard flow cytometry of whole blood samples?
- SSC (side scatter)
- FSC (forward scatter) (Correct answer)
- A fluorescence channel
- Time
Correct answer: FSC (forward scatter)
FSC-based triggering is standard for most applications because cell size (forward scatter) effectively distinguishes cellular events from electronic noise.
Question 4: A researcher is comparing two experiments run on different days and finds the MFI of a common marker differs by 30%. The best strategy to enable cross-experiment comparison is:
- Re-acquire all samples on the same day
- Use standardized reference beads to normalize instrument settings and MFI values across runs (Correct answer)
- Apply a fixed 30% correction factor
- Increase PMT voltage on the second run
Correct answer: Use standardized reference beads to normalize instrument settings and MFI values across runs
Calibration beads with known fluorochrome intensities enable instrument-independent normalization, allowing MFI comparisons across different days and instruments.
Question 5: In Boolean gating analysis, what does the gate 'CD3+ AND CD4+ AND NOT CD8+' identify?
- All T cells
- Helper T cells (CD4 single-positive) (Correct answer)
- Cytotoxic T cells
- Regulatory T cells
Correct answer: Helper T cells (CD4 single-positive)
CD3+CD4+CD8− defines conventional CD4 helper T cells, which express CD3 and CD4 but not CD8.
Question 6: When performing cell cycle analysis with propidium iodide (PI), why are cells typically treated with RNase before staining?
- To improve cell permeabilization
- Because PI intercalates into RNA as well as DNA, and RNase removes RNA to ensure only DNA content is measured (Correct answer)
- To fix cell morphology
- To reduce autofluorescence
Correct answer: Because PI intercalates into RNA as well as DNA, and RNase removes RNA to ensure only DNA content is measured
PI binds stoichiometrically to both DNA and RNA; RNase digestion removes RNA so that PI fluorescence reflects only DNA content for accurate cell cycle distribution.
Question 7: A flow cytometer's dynamic range is BEST described as:
- The maximum number of events it can collect per second
- The ratio between the dimmest and brightest signal the instrument can accurately measure and distinguish (Correct answer)
- The number of lasers available
- The range of cell sizes detectable by FSC
Correct answer: The ratio between the dimmest and brightest signal the instrument can accurately measure and distinguish
Dynamic range defines how many orders of magnitude of signal the system can resolve, which determines how well dim and bright populations can be simultaneously analyzed.
SCYM Data Acquisition & Analysis 5
In mass cytometry (CyTOF), heavy metal isotopes replace fluorochromes.
Which statement best describes why spillover is minimized in CyTOF?