SCYM Sample Preparation & Staining Techniques 2 — Questions and Answers
Question 1: Which fixative is most commonly used to preserve cell surface antigens for flow cytometric immunophenotyping?
- Glutaraldehyde 2.5%
- Paraformaldehyde 1-4% (Correct answer)
- Methanol 100%
- Acetone 100%
Correct answer: Paraformaldehyde 1-4%
Paraformaldehyde (1-4%) is preferred for surface antigen preservation because it cross-links proteins while maintaining epitope integrity better than stronger fixatives.
Question 2: When preparing peripheral blood for flow cytometry, which lysis method is preferred for maintaining lymphocyte scatter characteristics?
- Mechanical lysis by vortexing
- Ammonium chloride-based red cell lysis (Correct answer)
- Distilled water hypotonic lysis
- Saponin detergent lysis
Correct answer: Ammonium chloride-based red cell lysis
Ammonium chloride-based lysis selectively lyses red blood cells while preserving lymphocyte morphology and scatter properties.
Question 3: What is the primary purpose of blocking with normal serum prior to antibody staining in flow cytometry?
- To increase non-specific binding for better signal
- To saturate Fc receptors and reduce non-specific antibody binding (Correct answer)
- To fix the cells in suspension
- To permeabilize the cell membrane
Correct answer: To saturate Fc receptors and reduce non-specific antibody binding
Blocking with normal serum saturates Fc receptors on monocytes and other cells, preventing non-specific antibody binding that would produce false-positive results.
Question 4: Which parameter should be used to gate out dead cells when no viability dye is available?
- High forward scatter (FSC-H)
- Low side scatter (SSC-A)
- Low forward scatter and high side scatter (Correct answer)
- High CD45 expression
Correct answer: Low forward scatter and high side scatter
Dead cells characteristically show decreased FSC (cell shrinkage) and increased SSC (increased granularity from cellular debris), allowing them to be excluded by scatter gating.
Question 5: When performing intracellular cytokine staining, why is stimulation with brefeldin A or monensin required?
- To increase cell surface expression of cytokine receptors
- To block protein transport and accumulate cytokines inside cells (Correct answer)
- To permeabilize the plasma membrane
- To fix cytokines to antibodies
Correct answer: To block protein transport and accumulate cytokines inside cells
Brefeldin A and monensin block the Golgi apparatus, preventing secretion so that newly synthesized cytokines accumulate intracellularly and can be detected.
Question 6: Which detergent is most commonly used for cell permeabilization before intracellular antibody staining?
- SDS (sodium dodecyl sulfate)
- Saponin or Triton X-100 (Correct answer)
- Tween-80
- CHAPS
Correct answer: Saponin or Triton X-100
Saponin and Triton X-100 are the standard detergents for permeabilization, creating transient pores that allow antibody access without destroying cell morphology.
Question 7: A researcher notices that cells stained with a PE-conjugated antibody show high background fluorescence in the PE channel. What is the most likely cause?
- Too many cells in the sample
- Non-specific PE binding due to inadequate blocking or aggregated antibody (Correct answer)
- Incorrect laser selection for PE excitation
- Over-fixation with paraformaldehyde
Correct answer: Non-specific PE binding due to inadequate blocking or aggregated antibody
High PE background is most commonly caused by aggregated antibody or inadequate Fc blocking, leading to non-specific binding that appears as elevated signal.
SCYM Sample Preparation & Staining Techniques 2
Which fixative is most commonly used to preserve cell surface antigens for flow cytometric immunophenotyping?