ISAC Sample Preparation and Handling 2 — Questions and Answers
Question 1: Which fixative is most commonly used to preserve cell surface antigen integrity while allowing subsequent intracellular staining?
- Glutaraldehyde
- Paraformaldehyde (Correct answer)
- Methanol
- Acetone
Correct answer: Paraformaldehyde
Paraformaldehyde (PFA) at 1-4% preserves surface antigens and cell morphology while still permitting permeabilization for intracellular staining.
Question 2: When preparing peripheral blood mononuclear cells (PBMCs) via density gradient centrifugation, what is the primary purpose of the wash steps after isolation?
- To activate the lymphocytes before staining
- To remove platelets and residual gradient medium (Correct answer)
- To lyse any remaining red blood cells
- To increase cell concentration before staining
Correct answer: To remove platelets and residual gradient medium
Post-isolation washes remove platelets and residual Ficoll/gradient medium, both of which can interfere with antibody staining and instrument performance.
Question 3: A researcher notices high background fluorescence after staining frozen tissue sections for flow cytometry. Which blocking agent is most likely to reduce nonspecific antibody binding?
- BSA (bovine serum albumin)
- Saponin
- Normal serum from the host species of the secondary antibody (Correct answer)
- Tween-20
Correct answer: Normal serum from the host species of the secondary antibody
Normal serum from the same host species as the secondary antibody saturates nonspecific Fc receptor binding sites, reducing background staining.
Question 4: Which enzyme is most frequently used to dissociate solid tumors into single-cell suspensions for flow cytometric analysis?
- Hyaluronidase
- Trypsin
- Collagenase (Correct answer)
- Papain
Correct answer: Collagenase
Collagenase digests collagen fibers in the extracellular matrix, making it the preferred enzyme for dissociating solid tumors while maintaining surface antigen integrity.
Question 5: What is the recommended maximum time between blood collection and staining for surface markers when using whole blood protocols?
- 1 hour
- 4 hours
- 8 hours (Correct answer)
- 24 hours
Correct answer: 8 hours
Whole blood samples should ideally be stained within 8 hours of collection to minimize cellular activation, antigen degradation, and changes in leukocyte subsets.
Question 6: Which of the following is the best practice for storing antibody-stained samples that cannot be acquired immediately on the flow cytometer?
- Room temperature in the dark for up to 24 hours
- 4°C in the dark for up to 24 hours after fixation (Correct answer)
- −80°C freezer after adding DMSO
- 37°C incubator to maintain cell viability
Correct answer: 4°C in the dark for up to 24 hours after fixation
After fixation with PFA, samples can be stored at 4°C in the dark for up to 24 hours with minimal fluorochrome degradation or antigen loss.
Question 7: When dissociating lymph nodes for flow cytometry, mechanical disruption through a 70 µm cell strainer is preferred over enzymatic digestion primarily because:
- Enzymatic digestion is too expensive for routine use
- Mechanical methods preserve chemokine receptor expression better (Correct answer)
- Cell strainers yield higher total cell numbers
- Enzymes require biosafety level 3 containment
Correct answer: Mechanical methods preserve chemokine receptor expression better
Many chemokine receptors (e.g., CXCR4, CCR7) are proteolytically sensitive and are cleaved by enzymes like trypsin or collagenase, making mechanical disruption preferable.
ISAC Sample Preparation and Handling 2
Which fixative is most commonly used to preserve cell surface antigen integrity while allowing subsequent intracellular staining?