ISAC Analytical Methods and Techniques 3 — Questions and Answers
Question 1: What is the purpose of an FMO (Fluorescence Minus One) control in multicolor flow cytometry?
- To measure instrument background fluorescence only
- To define gate boundaries accounting for spectral spillover from all other channels (Correct answer)
- To calibrate laser power settings
- To normalize cell counts between samples
Correct answer: To define gate boundaries accounting for spectral spillover from all other channels
FMO controls contain all fluorochromes except the one being gated, revealing the true spread of spillover signal into that channel to set accurate gate boundaries.
Question 2: During cell sorting, what is the function of the piezoelectric crystal in the sort stream?
- It generates the electric charge on droplets
- It oscillates to break the fluid stream into uniform droplets (Correct answer)
- It deflects charged droplets into collection tubes
- It measures droplet velocity
Correct answer: It oscillates to break the fluid stream into uniform droplets
The piezoelectric crystal vibrates at a defined frequency to break the fluid stream into uniformly sized droplets at a precise break-off point.
Question 3: Which gating strategy best reduces doublet contamination in cell cycle analysis?
- Gating on FSC-A vs FSC-H or FSC-W (Correct answer)
- Gating on SSC-A vs SSC-W
- Using a DNA dye threshold gate only
- Applying a size exclusion filter before staining
Correct answer: Gating on FSC-A vs FSC-H or FSC-W
Plotting FSC-A (area) vs FSC-H (height) exploits the fact that doublets produce disproportionately high area signals, enabling their exclusion.
Question 4: In intracellular cytokine staining (ICS), why is brefeldin A or monensin used?
- To permeabilize the cell membrane for antibody entry
- To block protein secretion and retain cytokines inside cells (Correct answer)
- To fix cellular morphology before staining
- To enhance antibody binding affinity
Correct answer: To block protein secretion and retain cytokines inside cells
Brefeldin A and monensin block Golgi-mediated protein secretion, causing cytokines to accumulate intracellularly where they can be detected by flow cytometry.
Question 5: What statistical measure is most appropriate for describing the fluorescence intensity distribution of a heterogeneous cell population?
- Mean fluorescence intensity (MFI)
- Geometric mean fluorescence intensity (Correct answer)
- Mode
- Standard deviation only
Correct answer: Geometric mean fluorescence intensity
Geometric mean is preferred for log-scale fluorescence data because it accurately represents the central tendency of log-normal distributions without bias from extreme values.
Question 6: Which reagent is most commonly used to permeabilize cells for intracellular staining without disrupting surface marker expression?
- 0.1% Triton X-100
- Saponin-based buffers (Correct answer)
- 70% methanol
- Acetone
Correct answer: Saponin-based buffers
Saponin selectively permeabilizes cholesterol-rich membranes in a reversible manner, preserving surface epitopes better than detergents like Triton X-100.
Question 7: In index sorting, what additional data is collected for each sorted cell?
- DNA content at time of sort
- The fluorescence parameter values of that specific cell at time of sort (Correct answer)
- Cell cycle phase determination
- Viability status post-sort
Correct answer: The fluorescence parameter values of that specific cell at time of sort
Index sorting records the exact fluorescence intensity values for every parameter of each individual sorted cell, linking phenotypic data to downstream single-cell analysis.
ISAC Analytical Methods and Techniques 3
What is the purpose of an FMO (Fluorescence Minus One) control in multicolor flow cytometry?