Principles and applications of flow cytometry MCQs With Answer

Introduction: Flow cytometry is a powerful analytical technique widely used in cellular and molecular pharmacology to analyze physical and chemical characteristics of single cells or particles. This blog provides focused multiple-choice questions on the principles and applications of flow cytometry tailored for M.Pharm students. Questions cover instrumentation (optics, lasers, detectors), data parameters (forward and side scatter), fluorochrome selection, compensation and spectral unmixing, sample preparation, common assays (immunophenotyping, apoptosis, cell cycle), calibration, quality controls, and translational applications such as drug uptake and biomarker quantification. These MCQs reinforce conceptual understanding and practical considerations necessary for designing and interpreting flow cytometry experiments in pharmacological research.

Q1. What is the basic physical principle on which flow cytometry measures individual cells?

• Measurement of electrical impedance changes as cells pass through an aperture
• Measurement of scattered light and fluorescence from single cells in a hydrodynamically focused stream
• Imaging cells on a slide using transmitted light microscopy
• Measuring cell adhesion strength to a substrate

Correct Answer: Measurement of scattered light and fluorescence from single cells in a hydrodynamically focused stream

Q2. In flow cytometry, which parameter is most commonly correlated with cell size and which with internal complexity/granularity?

• Forward scatter correlates with granularity; side scatter correlates with size
• Forward scatter correlates with size; side scatter correlates with internal complexity/granularity
• Fluorescence correlates with size; forward scatter correlates with DNA content
• Side scatter correlates with viability; fluorescence correlates with cell count

Correct Answer: Forward scatter correlates with size; side scatter correlates with internal complexity/granularity

Q3. What does compensation accomplish in multicolor flow cytometry?

• It physically separates cells into different tubes based on fluorescence
• It adjusts lasers to emit at different wavelengths
• It mathematically corrects for spectral overlap (spillover) between fluorochromes
• It increases fluorescence intensity of dim fluorochromes

Correct Answer: It mathematically corrects for spectral overlap (spillover) between fluorochromes

Q4. What distinguishes FACS (fluorescence-activated cell sorting) from analytical flow cytometry?

• FACS uses only forward scatter to analyze cells
• FACS performs fluorescence measurement and physically sorts single cells by deflecting charged droplets
• FACS measures electrical impedance rather than fluorescence
• FACS cannot be used for live cells

Correct Answer: FACS performs fluorescence measurement and physically sorts single cells by deflecting charged droplets

Q5. What is the primary purpose of using an isotype control antibody in flow cytometry?

• To increase fluorochrome brightness
• To determine instrument voltage settings
• To estimate nonspecific antibody binding/background staining due to Fc or non-specific interactions
• To serve as a viability dye

Correct Answer: To estimate nonspecific antibody binding/background staining due to Fc or non-specific interactions

Q6. What does MESF (Molecules of Equivalent Soluble Fluorochrome) standardization quantify in flow cytometry?

• Cell concentration per milliliter
• Absolute number of fluorochrome molecules on a cell as a standardized fluorescence unit
• The side scatter intensity relative to forward scatter
• The number of cells sorted per second

Correct Answer: Absolute number of fluorochrome molecules on a cell as a standardized fluorescence unit

Q7. In apoptosis assays by flow cytometry, what does Annexin V binding indicate?

• Loss of mitochondrial membrane potential
• Externalization of phosphatidylserine on the outer leaflet of the plasma membrane (an early apoptosis marker)
• DNA fragmentation detected by sub-G1 peak
• Cell proliferation based on Ki-67 expression

Correct Answer: Externalization of phosphatidylserine on the outer leaflet of the plasma membrane (an early apoptosis marker)

Q8. Which controls are essential to perform accurate compensation in a multicolor experiment?

• Only an unstained control is required
• Only fluorescence minus one (FMO) controls are required
• Single-stained compensation controls (ideally using the same fluorochrome–antibody conjugates or beads) and an unstained control
• Only isotype controls are required for compensation

Correct Answer: Single-stained compensation controls (ideally using the same fluorochrome–antibody conjugates or beads) and an unstained control

Q9. How does spectral flow cytometry differ from conventional (bandpass-filter) flow cytometry?

• Spectral cytometry detects only forward and side scatter, not fluorescence
• Spectral cytometry collects the full emission spectrum across many detectors and uses unmixing algorithms to separate fluorochromes
• Spectral cytometry uses only one laser wavelength
• Spectral cytometry cannot be compensated mathematically

Correct Answer: Spectral cytometry collects the full emission spectrum across many detectors and uses unmixing algorithms to separate fluorochromes

Q10. Which parameter is most appropriate for assessing cell cycle distribution by flow cytometry?

• Surface CD markers expression
• DNA content measured by intercalating dyes such as propidium iodide (PI) or DAPI
• Side scatter representing granularity
• Annexin V binding alone

Correct Answer: DNA content measured by intercalating dyes such as propidium iodide (PI) or DAPI

Q11. What is meant by spillover spreading in multicolor flow cytometry?

• A reduction in cell size measured by forward scatter
• An increase in measurement variance (noise) in a parameter caused by spillover and compensation, degrading resolution of dim populations
• Improved separation of bright populations after compensation
• Physical mixing of fluorochromes inside a cell

Correct Answer: An increase in measurement variance (noise) in a parameter caused by spillover and compensation, degrading resolution of dim populations

Q12. Why are viability dyes (e.g., 7-AAD, DAPI, live/dead fixable dyes) used in flow cytometry experiments?

• To stain cell surface proteins more intensely
• To exclude dead cells that can bind antibodies nonspecifically and confound results
• To increase forward scatter signal
• To calibrate laser alignment

Correct Answer: To exclude dead cells that can bind antibodies nonspecifically and confound results

Q13. What is a bead-based multiplex assay in the context of flow cytometry?

• Using polystyrene beads to physically sort cells
• Using beads with distinct fluorescence or size signatures coupled to different capture antibodies to measure multiple soluble analytes simultaneously
• Using beads to block Fc receptors before staining
• Using beads to measure forward scatter calibration only

Correct Answer: Using beads with distinct fluorescence or size signatures coupled to different capture antibodies to measure multiple soluble analytes simultaneously

Q14. In flow cytometry analysis, what is “gating”?

• Setting the laser power to maximum
• Selecting subpopulations of interest in one or more parameter plots to include or exclude events for analysis
• Physically trapping cells in the flow cell
• Mixing cells with calibration beads

Correct Answer: Selecting subpopulations of interest in one or more parameter plots to include or exclude events for analysis

Q15. What role do photomultiplier tubes (PMTs) play in flow cytometers?

• They hydrodynamically focus the sample
• They detect fluorescent photons and amplify the signal to create an electrical output proportional to fluorescence intensity
• They sort cells by applying charges
• They produce the laser excitation light

Correct Answer: They detect fluorescent photons and amplify the signal to create an electrical output proportional to fluorescence intensity

Q16. How can cellular autofluorescence affect flow cytometry measurements?

• Autofluorescence enhances signal-to-noise ratio for all dyes
• Autofluorescence contributes background signal that can mask dim specific fluorescence and complicate panel design
• Autofluorescence only affects scatter parameters, not fluorescence
• Autofluorescence is eliminated by compensation automatically

Correct Answer: Autofluorescence contributes background signal that can mask dim specific fluorescence and complicate panel design

Q17. Which control is best when using tandem dyes that can degrade or change emission characteristics?

• Use only unstained cells
• Use single-stained compensation controls prepared with the same tandem-conjugated antibody and ideally on the same cell type or beads
• Use isotype controls only
• No special controls are needed for tandems

Correct Answer: Use single-stained compensation controls prepared with the same tandem-conjugated antibody and ideally on the same cell type or beads

Q18. Which of the following is a practical limitation of flow cytometry in pharmacological studies?

• It cannot analyze more than one parameter per cell
• Lack of spatial information (no tissue architecture) and requirement for single-cell suspensions that may alter biology
• It provides only qualitative data and never quantitative
• It is unsuitable for high-throughput analyses

Correct Answer: Lack of spatial information (no tissue architecture) and requirement for single-cell suspensions that may alter biology

Q19. Which application of flow cytometry is directly relevant to drug discovery and pharmacology?

• Measuring soil nutrient levels using fluorescent dyes
• Quantifying cell-surface receptor expression, transporter activity, drug uptake/efflux, and pharmacodynamic biomarker responses at single-cell resolution
• Imaging subcellular localization with high spatial resolution
• Measuring tissue elasticity in situ

Correct Answer: Quantifying cell-surface receptor expression, transporter activity, drug uptake/efflux, and pharmacodynamic biomarker responses at single-cell resolution

Q20. What is the purpose of monitoring the “time” parameter during flow cytometry acquisition?

• To measure the duration of laser emission pulses
• To detect acquisition instability, clogs, or drift over the run and allow gating out anomalous events collected during those periods
• To calculate cell doubling time from a single acquisition
• To replace the need for forward scatter measurements

Correct Answer: To detect acquisition instability, clogs, or drift over the run and allow gating out anomalous events collected during those periods

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