SCYM Flow Cytometry Principles 2 — Questions and Answers
Question 1: What is the primary function of the sheath fluid in a flow cytometer?
- To lyse red blood cells before analysis
- To hydrodynamically focus cells into a single-file stream (Correct answer)
- To stain cells with fluorescent dyes
- To cool the laser during operation
Correct answer: To hydrodynamically focus cells into a single-file stream
Sheath fluid surrounds the sample core and uses hydrodynamic focusing to align cells single-file through the laser interrogation point.
Question 2: Which scatter parameter is most useful for discriminating between cell types based on internal complexity?
- Forward scatter (FSC)
- Side scatter (SSC) (Correct answer)
- Fluorescence channel 1 (FL1)
- Time of flight
Correct answer: Side scatter (SSC)
Side scatter (SSC) is proportional to internal granularity and complexity, making it useful for distinguishing cell types like granulocytes from lymphocytes.
Question 3: In flow cytometry, what does 'compensation' correct for?
- Laser power fluctuations between runs
- Spectral overlap of fluorochrome emissions into adjacent detector channels (Correct answer)
- Background autofluorescence from unlabeled cells
- Sample viscosity differences between specimens
Correct answer: Spectral overlap of fluorochrome emissions into adjacent detector channels
Compensation mathematically subtracts the spillover signal of one fluorochrome detected in channels intended for other fluorochromes.
Question 4: What is the purpose of using single-stained controls in flow cytometry?
- To set the voltage for the FSC detector
- To calculate compensation values for spectral spillover (Correct answer)
- To determine the viability of cells before staining
- To calibrate the fluidics system pressure
Correct answer: To calculate compensation values for spectral spillover
Single-stained controls contain only one fluorochrome and are used to measure spillover into other channels for accurate compensation.
Question 5: Which of the following best describes 'gating' in flow cytometry data analysis?
- Filtering the sheath fluid to remove particles
- Defining a population of interest based on scatter or fluorescence parameters (Correct answer)
- Adjusting laser power to optimize signal
- Removing debris by centrifugation before acquisition
Correct answer: Defining a population of interest based on scatter or fluorescence parameters
Gating involves drawing boundaries on scatter plots or histograms to select specific cell populations for further analysis.
Question 6: The term 'fluorescence minus one' (FMO) control is used to:
- Measure non-specific antibody binding
- Determine the boundary between positive and negative populations in a multicolor panel (Correct answer)
- Assess laser alignment and beam shape
- Compensate for autofluorescence in fixed cells
Correct answer: Determine the boundary between positive and negative populations in a multicolor panel
FMO controls include all fluorochromes except one, revealing where the negative population boundary falls due to spectral spread from other channels.
Question 7: What happens to forward scatter (FSC) signal as cell size increases?
- FSC signal decreases proportionally
- FSC signal increases proportionally (Correct answer)
- FSC signal remains constant regardless of size
- FSC signal becomes negative for very large cells
Correct answer: FSC signal increases proportionally
Forward scatter is proportional to cell size; larger cells diffract more light in the forward direction, producing a stronger FSC signal.
SCYM Flow Cytometry Principles 2
What is the primary function of the sheath fluid in a flow cytometer?