LST Proliferation Assays 3

Question 1

Why is the tritiated thymidine pulse typically added only during the final 18–24 hours of a 5–6 day LST culture rather than at the beginning?

Accepted Answer

To capture peak S-phase activity without saturating the cells or depleting the label during early low-level proliferation

Answer

To reduce radiation exposure to laboratory personnel during the culture period

Answer

Because thymidine inhibits antigen presentation when present from day 0

Answer

To allow cytokine production to occur before DNA synthesis is measured

Question 2

When setting CFSE gates in flow cytometric analysis of a proliferation assay, the undivided parent population is identified by:

Accepted Answer

The highest fluorescence peak corresponding to cells that never divided and retained full CFSE label

Answer

The lowest fluorescence peak appearing after maximum dilution of the dye

Answer

Side scatter properties rather than fluorescence intensity

Answer

CD3 expression gated independently of CFSE signal

Question 3

Which of the following modifications optimizes a tritiated thymidine assay for detecting recall antigen responses, which are typically weaker than mitogen responses?

Accepted Answer

Extending culture time to 6–7 days and using higher antigen concentrations within non-toxic ranges

Answer

Reducing culture time to 24 hours to prevent antigen degradation

Answer

Omitting the negative control wells to increase throughput

Answer

Replacing ³H-thymidine with ¹⁴C-thymidine for higher energy emission

Question 4

In a Ki-67 staining-based proliferation assay, Ki-67 is a useful marker because it is expressed:

Accepted Answer

In all active phases of the cell cycle (G1, S, G2, M) but not in resting G0 cells

Answer

Only during S phase when DNA is being replicated

Answer

On the cell surface of all lymphocyte subsets regardless of proliferative status

Answer

Exclusively during mitosis in the M phase

Question 5

How does cell density at the time of seeding affect the outcome of a proliferation assay?

Accepted Answer

Too high a density can suppress proliferation due to contact inhibition and nutrient depletion, while too low a density reduces necessary paracrine cytokine signaling

Answer

Higher density always improves proliferation by maximizing cell-to-cell contact

Answer

Cell density has no effect on proliferation assay outcomes

Answer

Lower density always produces higher Stimulation Index values regardless of stimulant

Question 6

Which proliferation assay method allows simultaneous identification of the lymphocyte subsets (e.g., CD4+ vs. CD8+) that are proliferating in response to a stimulus?

Accepted Answer

CFSE dilution assay analyzed by multi-parameter flow cytometry with co-staining for surface markers

Answer

Tritiated thymidine incorporation with scintillation counting

Answer

WST-1 colorimetric assay

Answer

Total protein BCA assay measuring increased cell mass

Question 7

A laboratory observes that their LST proliferation assay consistently shows low Stimulation Index values with phytohemagglutinin (PHA) despite using standard protocols. The most likely cause is:

Accepted Answer

Compromised cell viability due to improper sample handling, prolonged transport time, or excessive freeze-thaw cycles

Answer

The use of too high a PHA concentration causing cell death

Answer

Overly long culture periods diluting the ³H-thymidine signal

Answer

Failure to add antigen to the positive control wells