ISAC Sample Preparation and Handling 3 ā Questions and Answers
Question 1: A viability dye that is excluded from live cells but penetrates dead cells with compromised membranes is classified as:
- A fixable viability dye
- A membrane-impermeant viability dye (Correct answer)
- A mitochondrial membrane potential dye
- An annexin-based apoptosis marker
Correct answer: A membrane-impermeant viability dye
Membrane-impermeant dyes like propidium iodide or 7-AAD rely on exclusion from intact membranes, staining only dead or dying cells with compromised membranes.
Question 2: Which centrifugation speed and time is most appropriate for pelleting lymphocytes without damaging cell membranes?
- 200 Ć g for 5 minutes
- 400 Ć g for 10 minutes (Correct answer)
- 1,200 Ć g for 15 minutes
- 3,000 Ć g for 5 minutes
Correct answer: 400 Ć g for 10 minutes
300ā400 Ć g for 8ā10 minutes is the standard range for gentle lymphocyte pelleting that avoids mechanical damage while achieving adequate recovery.
Question 3: When collecting bone marrow for flow cytometric immunophenotyping, why is it important to use EDTA rather than heparin as an anticoagulant?
- EDTA prevents bacterial contamination during processing
- Heparin can cause non-specific binding that interferes with CD34 staining (Correct answer)
- EDTA preserves RNA integrity better than heparin
- Heparin activates platelets that clog the flow cytometer
Correct answer: Heparin can cause non-specific binding that interferes with CD34 staining
Heparin can bind to CD34 and other hematopoietic progenitor antigens, causing non-specific staining artifacts that complicate immunophenotyping of stem cell populations.
Question 4: The 'hook effect' in flow cytometry panels occurs when:
- Too many fluorochromes are used in a single panel
- Excess antibody saturates both specific and non-specific binding sites (Correct answer)
- The laser power is set too high for bright fluorochromes
- Cells are incubated at the wrong temperature during staining
Correct answer: Excess antibody saturates both specific and non-specific binding sites
The hook effect occurs when antibody is present in excess, leading to reduced mean fluorescence intensity (MFI) as excess antibody competes with bound antibody.
Question 5: For optimal staining of cytokines using intracellular flow cytometry, cells must be treated with a protein transport inhibitor such as brefeldin A or monensin. What is the purpose of this step?
- To fix the cells before permeabilization
- To block cytokine secretion so cytokines accumulate intracellularly (Correct answer)
- To enhance antibody penetration through the cell membrane
- To prevent non-specific antibody binding in the cytoplasm
Correct answer: To block cytokine secretion so cytokines accumulate intracellularly
Brefeldin A and monensin block the Golgi apparatus secretory pathway, causing cytokines to accumulate intracellularly where they can be detected by antibody staining.
Question 6: When preparing single-cell suspensions from lung tissue for flow cytometry, what is a major concern with enzymatic digestion protocols?
- Over-digestion can destroy surface epitopes critical for phenotyping (Correct answer)
- Enzymes introduce autofluorescence that overwhelms detector signals
- Enzymatic digestion generates too many doublets for accurate gating
- Lung enzymes are too expensive for routine laboratory use
Correct answer: Over-digestion can destroy surface epitopes critical for phenotyping
Prolonged enzymatic digestion can cleave surface epitopes (e.g., CD11c, SiglecF on alveolar macrophages), leading to false-negative results in phenotyping panels.
Question 7: Which of the following best describes the purpose of an Fc receptor block (e.g., Human TruStain FcX) in flow cytometry staining protocols?
- It prevents cell aggregation during incubation
- It inhibits non-specific antibody binding via Fc regions to Fc receptors on immune cells (Correct answer)
- It blocks intracellular Fc receptors from binding secondary antibodies
- It reduces autofluorescence from dead cells
Correct answer: It inhibits non-specific antibody binding via Fc regions to Fc receptors on immune cells
Fc receptor blocking reagents saturate FcĪ³R (CD16, CD32, CD64) on monocytes, NK cells, and granulocytes, preventing non-specific antibody binding through the Fc region rather than the antigen-binding domain.
ISAC Sample Preparation and Handling 3
A viability dye that is excluded from live cells but penetrates dead cells with compromised membranes is classified as: