LST Proliferation Assays 2 — Questions and Answers
Question 1: In CFSE dilution assays analyzed by flow cytometry, each successive generation of dividing lymphocytes is characterized by:
- Fluorescence intensity halved relative to the previous generation (Correct answer)
- Fluorescence intensity doubled relative to the previous generation
- No change in fluorescence but increased cell size
- Increased forward scatter with constant fluorescence
Correct answer: Fluorescence intensity halved relative to the previous generation
Because CFSE is equally partitioned between two daughter cells at each division, each generation displays fluorescence that is exactly half that of the parent generation, creating discrete peaks on a histogram.
Question 2: The Proliferation Index (PI) in a CFSE assay differs from the Division Index in that PI measures:
- The average number of divisions undergone by cells that actually divided (Correct answer)
- The fraction of total cells that entered at least one division
- The total number of cells produced from the original population
- The percentage of cells remaining undivided
Correct answer: The average number of divisions undergone by cells that actually divided
The Proliferation Index is the average number of divisions per dividing cell (excludes undivided cells from the calculation), whereas the Division Index includes undivided cells in the denominator.
Question 3: Which cell density is generally optimal for seeding peripheral blood mononuclear cells (PBMCs) in a standard 96-well proliferation assay?
- 1–2 × 10⁵ cells per well (Correct answer)
- 1–2 × 10³ cells per well
- 1–2 × 10⁷ cells per well
- 5 × 10⁴ cells per well in 10 mL medium
Correct answer: 1–2 × 10⁵ cells per well
A seeding density of 1–2 × 10⁵ PBMCs per well in a 96-well plate provides adequate cell-to-cell contact for co-stimulation while avoiding overcrowding that suppresses proliferation or depletes nutrients.
Question 4: In a tritiated thymidine proliferation assay, cells are typically harvested onto glass fiber filters using a:
- Semi-automated cell harvester (Correct answer)
- Centrifuge and pipette aspiration
- Magnetic bead separation system
- Density gradient centrifugation
Correct answer: Semi-automated cell harvester
A semi-automated cell harvester lyses cells and deposits their DNA onto glass fiber filter mats, efficiently washing away unincorporated ³H-thymidine before scintillation counting.
Question 5: EdU (5-ethynyl-2'-deoxyuridine) offers a technical advantage over BrdU for proliferation assays primarily because:
- EdU detection does not require harsh DNA denaturation steps that damage cell surface antigens (Correct answer)
- EdU emits fluorescence without requiring antibody staining
- EdU is incorporated into RNA rather than DNA
- EdU detection can be performed without fixation
Correct answer: EdU detection does not require harsh DNA denaturation steps that damage cell surface antigens
EdU is detected via a click chemistry reaction that does not require the acid or heat denaturation needed to expose BrdU epitopes, thereby preserving cell surface markers and enabling robust multi-parameter flow cytometry.
Question 6: In an LST proliferation assay, what does a high background CPM in the unstimulated negative control most likely indicate?
- Spontaneous proliferation due to activated cells, contamination, or excessive cell death triggering bystander activation (Correct answer)
- Optimal assay conditions with well-functioning lymphocytes
- Adequate mitogen stimulation of the control well
- Correct thymidine pulse timing
Correct answer: Spontaneous proliferation due to activated cells, contamination, or excessive cell death triggering bystander activation
Elevated background CPM in negative controls suggests non-specific lymphocyte activation from microbial contamination, excessive cell stress, or the presence of in-vivo pre-activated lymphocytes, all of which compromise assay validity.
Question 7: The WST-1 colorimetric assay measures lymphocyte proliferation indirectly by detecting:
- Mitochondrial dehydrogenase activity as a surrogate for viable, metabolically active cells (Correct answer)
- Direct DNA synthesis via nucleoside incorporation
- Cytokine release into the culture supernatant
- Cell surface expression of proliferation markers like Ki-67
Correct answer: Mitochondrial dehydrogenase activity as a surrogate for viable, metabolically active cells
WST-1 is cleaved by mitochondrial dehydrogenases in metabolically active cells to produce a formazan dye; absorbance at 450 nm correlates with viable cell number, providing an indirect measure of proliferation.
LST Proliferation Assays 2
In CFSE dilution assays analyzed by flow cytometry, each successive generation of dividing lymphocytes is characterized by: