SCYM Sample Preparation & Staining Techniques 4 — Questions and Answers
Question 1: What is the rationale for titrating antibodies before use in a flow cytometry panel?
- To determine the maximum antibody dose that causes cell death
- To find the optimal concentration giving maximum signal-to-noise ratio (Correct answer)
- To test antibody stability at different temperatures
- To identify non-specific secondary antibody binding
Correct answer: To find the optimal concentration giving maximum signal-to-noise ratio
Antibody titration identifies the concentration that produces the highest staining index (signal-to-noise), preventing both under-staining and excess background from over-staining.
Question 2: Which method is used to preserve cytokine-secreting cells for intracellular cytokine staining when immediate processing is not possible?
- Storage in liquid nitrogen without cryoprotectant
- Fixation with methanol then storage at -80°C
- Addition of brefeldin A during stimulation then fixation with paraformaldehyde (Correct answer)
- Storage in PBS at 4°C for up to 5 days
Correct answer: Addition of brefeldin A during stimulation then fixation with paraformaldehyde
Adding brefeldin A during the stimulation period to trap cytokines, followed by paraformaldehyde fixation, allows samples to be safely stored until staining.
Question 3: Which fluorochrome combination is INCOMPATIBLE due to spectral overlap that cannot be adequately compensated?
- FITC and PE-Cy7
- PE and PE-Cy5 (Correct answer)
- APC and APC-H7
- BV421 and Pacific Blue
Correct answer: PE and PE-Cy5
PE and PE-Cy5 share the same PE excitation and have significant spectral overlap with incomplete energy transfer, making compensation problematic and results unreliable.
Question 4: When staining for phosphorylated signaling proteins by flow cytometry, which fixation protocol is typically required?
- Paraformaldehyde fixation followed by saponin permeabilization
- Methanol fixation (cold, -20°C) alone
- Glutaraldehyde fixation followed by Triton X-100
- Formaldehyde fixation followed by methanol permeabilization (Correct answer)
Correct answer: Formaldehyde fixation followed by methanol permeabilization
The BD Phosflow protocol uses formaldehyde fixation to cross-link proteins followed by cold methanol permeabilization, which preserves phosphoepitopes while allowing antibody access.
Question 5: A technician is preparing a whole-blood lysed sample and notices that the monocyte gate on FSC/SSC shows poor resolution. What is the most likely cause?
- Excess antibody concentration
- Incomplete red cell lysis leaving residual erythrocytes (Correct answer)
- Using an incorrect laser line
- Over-fixation causing cell swelling
Correct answer: Incomplete red cell lysis leaving residual erythrocytes
Residual unlysed red blood cells overlap with the monocyte population on FSC/SSC scatter plots, obscuring the distinct monocyte cloud.
Question 6: What is the purpose of including a fluorescence-minus-one (FMO) control in a multicolor panel?
- To compensate for spectral spillover between channels
- To set the positive/negative gate boundary accounting for spread from other fluorochromes (Correct answer)
- To measure non-specific antibody binding
- To calibrate the laser power
Correct answer: To set the positive/negative gate boundary accounting for spread from other fluorochromes
FMO controls contain all fluorochromes except the one being gated, revealing how spectral spillover from other channels affects the gate boundary in that channel.
Question 7: Which cell preparation technique is used to isolate peripheral blood mononuclear cells (PBMCs) prior to some flow cytometry assays?
- Red cell lysis with ammonium chloride
- Density gradient centrifugation using Ficoll-Hypaque (Correct answer)
- Magnetic bead positive selection for CD3
- Centrifugation at 800×g without gradient
Correct answer: Density gradient centrifugation using Ficoll-Hypaque
Ficoll-Hypaque density gradient centrifugation separates PBMCs (lymphocytes and monocytes) from granulocytes and red cells by buoyant density.
SCYM Sample Preparation & Staining Techniques 4
What is the rationale for titrating antibodies before use in a flow cytometry panel?