SCYM Cell Cycle & DNA Content Analysis 1 — Questions and Answers
Question 1: Which fluorescent dye is most commonly used for DNA content analysis in flow cytometry and requires RNase treatment for accurate results?
- DAPI
- Propidium Iodide (PI) (Correct answer)
- 7-AAD
- Hoechst 33342
Correct answer: Propidium Iodide (PI)
Propidium iodide intercalates into double-stranded nucleic acids including RNA, so RNase A treatment is required to eliminate RNA-derived signal and ensure fluorescence reflects only DNA content.
Question 2: In a DNA content histogram, which cell cycle phase is represented by the 4N peak?
- G0/G1 phase
- Early S phase
- G2 and M phase (Correct answer)
- Sub-G1 apoptotic fraction
Correct answer: G2 and M phase
Cells in G2 and M phases have completed DNA replication and contain exactly twice the diploid (2N) DNA content, producing the 4N peak in a DNA histogram.
Question 3: What does a low coefficient of variation (CV) of the G0/G1 peak in a DNA histogram indicate?
- High percentage of cells in S phase
- Uniform DNA staining and good assay quality (Correct answer)
- Presence of aneuploid cell populations
- Excessive cell doublets and aggregates
Correct answer: Uniform DNA staining and good assay quality
A low CV of the G0/G1 peak reflects homogeneous DNA staining, optimal sample preparation, and high instrument precision, indicating a high-quality DNA content assay.
Question 4: What is the DNA index (DI) of a normal diploid cell population used as a reference standard?
- 0.5
- 1.0 (Correct answer)
- 2.0
- 4.0
Correct answer: 1.0
The DNA index is the ratio of the G0/G1 peak position of the test cells to a diploid reference standard; normal diploid cells have a DI of exactly 1.0.
Question 5: Which cell cycle phase is characterized by active DNA synthesis, with cells containing DNA content between 2N and 4N?
- G0 (quiescence)
- G1 phase
- S phase (Correct answer)
- G2 phase
Correct answer: S phase
S phase (synthesis phase) is when the cell replicates its entire genome, resulting in a continuous distribution of DNA content between 2N and 4N that forms the S phase fraction in a histogram.
Question 6: Which DNA-binding dye can stain live cells without fixation because it is membrane-permeant?
- Propidium Iodide
- 7-AAD
- Hoechst 33342 (Correct answer)
- Ethidium Bromide
Correct answer: Hoechst 33342
Hoechst 33342 is a cell-permeant bisbenzimide dye that binds AT-rich regions of dsDNA in the minor groove and can enter live cells, enabling DNA content analysis without fixation.
Question 7: A sub-G1 peak in a DNA histogram is most commonly associated with which cellular process?
- Cell aggregation artifacts
- Apoptosis with DNA fragmentation (Correct answer)
- Necrotic cell lysis
- G1 phase arrest
Correct answer: Apoptosis with DNA fragmentation
The sub-G1 peak represents apoptotic cells in which internucleosomal DNA cleavage by caspase-activated DNase has reduced total DNA content below the 2N level.
