SCYM Sample Preparation & Staining Techniques 5 — Questions and Answers
Question 1: When staining cells for apoptosis using Annexin V/PI, what is the interpretation of Annexin V+/PI- cells?
- Necrotic cells with compromised membranes
- Early apoptotic cells with intact membranes (Correct answer)
- Live cells with high phosphatidylserine expression
- Late apoptotic or secondary necrotic cells
Correct answer: Early apoptotic cells with intact membranes
Annexin V+/PI- represents early apoptosis: phosphatidylserine is externalized (binding Annexin V) but the plasma membrane remains intact (excluding PI).
Question 2: Which condition is essential when performing Annexin V staining to ensure proper binding?
- Alkaline buffer at pH 9.0
- Calcium-containing binding buffer (Correct answer)
- EDTA to prevent cell aggregation
- Methanol fixation prior to staining
Correct answer: Calcium-containing binding buffer
Annexin V binding to phosphatidylserine is calcium-dependent; the staining buffer must contain physiological calcium concentrations (typically 2.5 mM CaCl₂).
Question 3: A researcher wants to measure proliferation of T cells using CFSE. Which statement correctly describes the assay principle?
- CFSE fluorescence increases with each cell division
- CFSE covalently labels proteins, and fluorescence halves with each cell division (Correct answer)
- CFSE is incorporated into newly synthesized DNA
- CFSE specifically labels the nucleus
Correct answer: CFSE covalently labels proteins, and fluorescence halves with each cell division
CFSE covalently binds intracellular proteins; at each division, the dye is distributed equally between daughter cells, so fluorescence intensity halves with each generation.
Question 4: What is the recommended storage condition for cells after paraformaldehyde fixation when same-day acquisition is not possible?
- Room temperature in the dark for up to 7 days
- 4°C protected from light for up to 24-72 hours (Correct answer)
- -80°C in cryoprotectant
- 37°C in complete media
Correct answer: 4°C protected from light for up to 24-72 hours
Fixed samples are stable at 4°C in the dark for 24-72 hours; longer storage can cause autofluorescence increases and epitope degradation.
Question 5: Which pre-analytical variable most significantly affects the absolute count of CD34+ hematopoietic progenitor cells in peripheral blood?
- Time of day the blood is drawn
- Anticoagulant used (EDTA vs. citrate)
- Sample temperature and time from collection to processing (Correct answer)
- Tube inversion technique
Correct answer: Sample temperature and time from collection to processing
CD34+ cells are particularly sensitive to delayed processing and temperature extremes; cells begin to die and expression levels change within a few hours if samples are not handled properly.
Question 6: When designing a multicolor panel, why should the brightest fluorochromes be paired with the lowest-expressed antigens?
- Bright fluorochromes have less spectral spillover
- Dim antigens need high signal to be resolved from background noise (Correct answer)
- Bright fluorochromes are more stable at low antigen densities
- Low-expressed antigens require less compensation
Correct answer: Dim antigens need high signal to be resolved from background noise
Low-density antigens generate few antibody-bound fluorochrome molecules per cell; using a bright fluorochrome maximizes the signal from these sparse targets, improving resolution from negative cells.
Question 7: Which enzyme is commonly used during tissue dissociation protocols to release cells from connective tissue matrix for subsequent flow cytometry?
- DNase I alone
- Collagenase (with or without hyaluronidase) (Correct answer)
- Proteinase K
- Trypsin-EDTA at high concentration
Correct answer: Collagenase (with or without hyaluronidase)
Collagenase digests the collagen-rich extracellular matrix in solid tissues, releasing cells with minimal damage to surface antigens, often combined with hyaluronidase to break down other ECM components.
SCYM Sample Preparation & Staining Techniques 5
When staining cells for apoptosis using Annexin V/PI, what is the interpretation of Annexin V+/PI- cells?