ISAC Data Analysis and Interpretation 3

Question 1

When analyzing cell cycle data stained with propidium iodide (PI), which cell cycle phase corresponds to the G2/M peak?

Accepted Answer

The peak with exactly twice the DNA content of G1

Answer

G2/M cells have undergone DNA replication and contain twice (4N) the DNA content of G1 (2N) cells, appearing as a distinct peak at double the G1 channel.

Question 2

In spectral flow cytometry, what is the key mathematical process that separates overlapping emission spectra into individual fluorochrome contributions?

Accepted Answer

Spectral unmixing using a reference library of pure spectra

Answer

Spectral unmixing uses least-squares fitting against a library of single-fluorochrome reference spectra to deconvolve overlapping signals across all detectors.

Question 3

A dot plot showing FSC-A on the X-axis and FSC-H on the Y-axis is PRIMARILY used to:

Accepted Answer

Identify cell doublets by discriminating cell aggregates from singlets

Answer

Doublet discrimination plots use FSC-A vs. FSC-H because doublets have proportionally larger area than height, falling off the diagonal of singlets.

Question 4

Which statistical measure is MOST appropriate for describing the central tendency of a highly skewed fluorescence distribution?

Accepted Answer

Median fluorescence intensity

Answer

Median is resistant to outliers and skewed distributions, making it more representative of the central tendency than mean for non-normal fluorescence data.

Question 5

In the context of SPADE (Spanning-tree Progression Analysis of Density-normalized Events), nodes in the tree represent:

Accepted Answer

Clusters of phenotypically similar cells

Answer

SPADE clusters cells into nodes of similar phenotype and connects them in a spanning tree that reflects developmental or phenotypic relationships.

(ISAC) International Society for the Advancement of Cytometry Practice Test

(ISAC) International Society for the Advancement of Cytometry Practice Test

ISAC Data Analysis and Interpretation 3