Principles and applications of flow cytometry MCQs With Answer

Introduction

This set of 20 targeted MCQs on the principles and applications of flow cytometry is designed for M.Pharm students studying Modern Bio-Analytical Techniques (MPA 202T). The questions cover core concepts — fluidics, optics, electronics, detectors, data processing, and multicolor panel design — and extend to practical applications such as immunophenotyping, cell cycle and viability assays, absolute counting, sorting, and nanoparticle analysis. Each item emphasizes analytical reasoning, instrument settings, troubleshooting, and experimental design relevant to pharmaceutical research and quality control. Use these questions to test understanding, prepare for exams, and sharpen skills needed for applying flow cytometry in drug development and biopharmaceutical characterization.

Q1. Which component of a flow cytometer is primarily responsible for hydrodynamically focusing cells into a single-file stream so they pass one-by-one through the interrogation point?

• Sheath fluid and flow cell arrangement
• Laser excitation system
• Photomultiplier tubes (PMTs)
• Analogue-to-digital converter (ADC)

Correct Answer: Sheath fluid and flow cell arrangement

Q2. Forward scatter (FSC) and side scatter (SSC) provide which types of information about cells?

• FSC indicates granularity and SSC indicates cell size
• FSC indicates cell size and SSC indicates internal complexity/granularity
• Both FSC and SSC measure fluorescence intensity
• FSC measures nucleic acid content and SSC measures membrane potential

Correct Answer: FSC indicates cell size and SSC indicates internal complexity/granularity

Q3. What is fluorescence compensation and why is it necessary in multicolor flow cytometry?

• Adjustment of laser power to equalize fluorophore brightness
• Mathematical correction for spectral overlap between fluorochromes to prevent false positives
• Normalization of cell counts across samples for accurate absolute counting
• Calibration of PMT voltages to reduce electronic noise

Correct Answer: Mathematical correction for spectral overlap between fluorochromes to prevent false positives

Q4. Which detector type is most commonly used to measure low-intensity fluorescence signals in conventional flow cytometers?

• Charge-coupled device (CCD) camera
• Photodiode
• Photomultiplier tube (PMT)
• Complementary metal-oxide-semiconductor (CMOS) sensor

Correct Answer: Photomultiplier tube (PMT)

Q5. In pulse processing, what does “height, area and width” refer to and how can width be used experimentally?

• They are parameters of electrical noise; width indicates instrument calibration status
• They describe fluorescence pulse shape; width can discriminate doublets from single cells
• They are measures of laser beam quality; height shows power and width shows divergence
• They quantify PMT gain; area is gain, height is offset, width is noise

Correct Answer: They describe fluorescence pulse shape; width can discriminate doublets from single cells

Q6. When designing a multicolor panel, which fluorochrome selection strategy reduces compensation complexity and improves resolution?

• Choosing fluorochromes with maximal spectral overlap for stronger signals
• Assigning brightest fluorochromes to least abundant or dim antigens and minimizing spectral overlap
• Using only tandem dyes to maximize channel usage
• Assigning all fluorochromes excited by a single laser to the most important markers

Correct Answer: Assigning brightest fluorochromes to least abundant or dim antigens and minimizing spectral overlap

Q7. What is the primary purpose of using calibration beads (fluorescent reference beads) before running experimental samples?

• To lyse red blood cells prior to analysis
• To standardize instrument sensitivity, align detectors, and allow quantitative comparison between runs
• To stain cells for viability assessment
• To calibrate fluidics pressure and sheath flow rate only

Correct Answer: To standardize instrument sensitivity, align detectors, and allow quantitative comparison between runs

Q8. Which control is essential to set gates for multicolor experiments and to evaluate contribution of autofluorescence and spillover from multiple fluorophores?

• Unstained sample and single-stain controls (including fluorescence minus one, FMO)
• Only fully stained sample without controls
• Only an isotype control for each fluorochrome
• Only a live/dead stain control

Correct Answer: Unstained sample and single-stain controls (including fluorescence minus one, FMO)

Q9. Which fixation/permeabilization approach is most appropriate for intracellular cytokine staining without disrupting surface marker detection?

• Strong organic solvents (methanol) only
• Paraformaldehyde fixation followed by a mild detergent-based permeabilization (e.g., saponin)
• Boiling samples for denaturation
• No fixation to preserve cytokines

Correct Answer: Paraformaldehyde fixation followed by a mild detergent-based permeabilization (e.g., saponin)

Q10. In absolute cell counting by flow cytometry using counting beads, what calculation principle is used to determine cell concentration?

• Ratio of live cells to dead cells multiplied by sample volume
• Ratio of counted sample events to counted bead events multiplied by known bead concentration and dilution factor
• Total events per second divided by flow rate speed
• Mean fluorescence intensity of beads compared to cells

Correct Answer: Ratio of counted sample events to counted bead events multiplied by known bead concentration and dilution factor

Q11. Spectral flow cytometry differs from conventional flow cytometry primarily because:

• It uses fewer lasers and only measures FSC and SSC
• It collects the entire emission spectrum from each particle and uses unmixing algorithms to separate fluorophores
• It cannot perform cell sorting due to spectral complexity
• It only measures autofluorescence and not labeled fluorophores

Correct Answer: It collects the entire emission spectrum from each particle and uses unmixing algorithms to separate fluorophores

Q12. What is the most likely cause if all fluorescence channels show an unusually high background signal after staining?

• Incorrect PMT voltage set too low
• High sample autofluorescence, contaminated buffers, or improper wash leading to unbound fluorochrome
• Cell doublet formation only
• Laser turned off

Correct Answer: High sample autofluorescence, contaminated buffers, or improper wash leading to unbound fluorochrome

Q13. For cell cycle analysis using DNA dyes like propidium iodide, which step is essential to obtain accurate DNA content measurements?

• Staining live cells without fixation to retain cell cycle proteins
• Permeabilization and RNase treatment to remove RNA interference with DNA signal
• Using viability dyes to exclude G0 cells only
• Using isotype controls to set gates

Correct Answer: Permeabilization and RNase treatment to remove RNA interference with DNA signal

Q14. During fluorescence-activated cell sorting (FACS), what parameter is most critical to ensure high purity of the sorted population?

• Sorting nozzle size and drop delay calibration aligned with gating strategy
• Using the highest possible sheath pressure irrespective of cell type
• Maximizing sample concentration to increase event rate
• Disabling compensation to simplify sorting

Correct Answer: Sorting nozzle size and drop delay calibration aligned with gating strategy

Q15. Which metric describes instrument resolution for detecting dim signals in flow cytometry and is often evaluated with fluorescent beads?

• Coefficient of variation (CV) of bead fluorescence peak
• Flow rate variability
• Number of lasers available
• Sheath fluid conductivity

Correct Answer: Coefficient of variation (CV) of bead fluorescence peak

Q16. In nanoparticle analysis by flow cytometry, why is side scatter representation often shifted to a logarithmic scale and instrument sensitivity critical?

• Particles are large and bright so linear scale is adequate
• Nanoparticles produce very low scatter signals spanning several orders of magnitude, requiring high sensitivity and logarithmic scaling for resolution
• Log scale hides noise and prevents detection of small particles
• Instrument sensitivity is irrelevant for nanoparticles detection

Correct Answer: Nanoparticles produce very low scatter signals spanning several orders of magnitude, requiring high sensitivity and logarithmic scaling for resolution

Q17. What is the best practice for titrating antibodies for flow cytometry panels to achieve optimal signal-to-noise ratio?

• Use manufacturer recommended concentration without further testing
• Perform serial dilution titration on representative cells to find the saturating concentration that gives maximal specific signal and minimal background
• Always use twice the recommended concentration to ensure brightest staining
• Use crude lysates to determine antibody amount

Correct Answer: Perform serial dilution titration on representative cells to find the saturating concentration that gives maximal specific signal and minimal background

Q18. What is fluorescence minus one (FMO) control used for in multicolor experiments?

• To determine the autofluorescence spectrum of the instrument only
• To define gating boundaries by including all fluorochromes except the one of interest, revealing spread from other markers
• To calibrate PMT voltages for each individual fluorochrome
• To replace unstained control when cell numbers are limited

Correct Answer: To define gating boundaries by including all fluorochromes except the one of interest, revealing spread from other markers

Q19. When analyzing rare cell populations (<0.1% of total events), which strategy improves detection reliability?

• Run a small volume quickly to avoid sample degradation
• Acquire a very large total event count, use enrichment protocols, and include appropriate controls to validate gates
• Reduce sample concentration to minimize coincidence only
• Exclude viability staining to save fluorochrome channels

Correct Answer: Acquire a very large total event count, use enrichment protocols, and include appropriate controls to validate gates

Q20. In data analysis, what does median fluorescence intensity (MFI) represent and why might it be preferred over mean fluorescence for skewed populations?

• MFI represents the average intensity of all channels and is sensitive to outliers
• MFI is the midpoint of the distribution and is less affected by extreme values, making it more robust for skewed populations
• MFI measures the total fluorescence per sample volume
• MFI reflects instrument noise rather than biological signal

Correct Answer: MFI is the midpoint of the distribution and is less affected by extreme values, making it more robust for skewed populations

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