LST Proliferation Assays 1 — Questions and Answers
Question 1: Which radioactive compound is most commonly used to measure DNA synthesis in the tritiated thymidine incorporation assay for lymphocyte proliferation?
- ³H-thymidine (Correct answer)
- ¹⁴C-uridine
- ³²P-phosphate
- ¹²⁵I-iododeoxyuridine
Correct answer: ³H-thymidine
³H-thymidine (tritiated thymidine) is incorporated into newly synthesized DNA during cell division, making it the gold standard radiometric marker for lymphocyte proliferation assays.
Question 2: In the tritiated thymidine incorporation assay, what instrument is used to quantify the radioactivity incorporated into lymphocyte DNA?
- Liquid scintillation counter (Correct answer)
- Gamma counter
- Flow cytometer
- Spectrophotometer
Correct answer: Liquid scintillation counter
A liquid scintillation counter is used to measure the beta emissions from ³H-thymidine incorporated into DNA, providing counts per minute (CPM) as a measure of proliferation.
Question 3: CFSE (carboxyfluorescein succinimidyl ester) measures lymphocyte proliferation by which mechanism?
- Progressive dilution of the dye with each cell division (Correct answer)
- Increased fluorescence intensity upon DNA synthesis
- Binding to newly expressed surface antigens
- Enzymatic cleavage releasing fluorescent product
Correct answer: Progressive dilution of the dye with each cell division
CFSE covalently labels intracellular proteins, and with each cell division the dye is equally distributed between daughter cells, halving fluorescence intensity and allowing enumeration of division cycles by flow cytometry.
Question 4: The Stimulation Index (SI) in an LST proliferation assay is calculated as:
- CPM of stimulated cells / CPM of unstimulated cells (Correct answer)
- CPM of unstimulated cells / CPM of stimulated cells
- (CPM stimulated − CPM unstimulated) × 100
- CPM stimulated − CPM background
Correct answer: CPM of stimulated cells / CPM of unstimulated cells
The Stimulation Index is the ratio of radioactive incorporation (or other proliferation signal) in antigen-stimulated cells divided by that in unstimulated control cells, with a ratio ≥ 2–3 generally considered positive.
Question 5: What is the typical duration of lymphocyte culture before adding tritiated thymidine in a standard LST proliferation assay?
- 5–6 days (Correct answer)
- 1–2 hours
- 24 hours
- 10–14 days
Correct answer: 5–6 days
Lymphocytes are cultured for 5–6 days to allow sufficient antigen processing, T-cell activation, and clonal expansion before a tritiated thymidine pulse is added in the final 18–24 hours.
Question 6: BrdU (bromodeoxyuridine) incorporation assays detect lymphocyte proliferation by measuring:
- BrdU incorporated into newly synthesized DNA using anti-BrdU antibodies (Correct answer)
- Fluorescence emitted directly by BrdU during UV excitation
- BrdU-induced changes in cell surface marker expression
- BrdU release from dying cells into culture supernatant
Correct answer: BrdU incorporated into newly synthesized DNA using anti-BrdU antibodies
BrdU is a thymidine analogue incorporated into DNA during S phase; proliferating cells are detected by staining with anti-BrdU antibodies after DNA denaturation, allowing analysis by flow cytometry or ELISA.
Question 7: Which of the following is a major safety advantage of CFSE-based and BrdU-based proliferation assays over the tritiated thymidine incorporation method?
- They do not require radioactive materials or generate radioactive waste (Correct answer)
- They are more sensitive at detecting low-level proliferation
- They require shorter incubation times
- They are less expensive per assay
Correct answer: They do not require radioactive materials or generate radioactive waste
CFSE and BrdU assays are non-radioactive, eliminating the need for radiation safety protocols, special licensing, and radioactive waste disposal required for ³H-thymidine assays.
LST Proliferation Assays 1
Which radioactive compound is most commonly used to measure DNA synthesis in the tritiated thymidine incorporation assay for lymphocyte proliferation?