SCYM Data Analysis 5

Question 1

In a proliferation assay using CFSE dilution, how should the data be analyzed to calculate division index?

Accepted Answer

Use peak-fitting software to enumerate cells in each division generation and calculate the mean divisions per original cell

Answer

Division index analysis requires fitting individual generation peaks and calculating the mean number of cell divisions undergone by all cells in the original population.

Question 2

Which quality control metric best evaluates the consistency of sample acquisition across multiple tubes in a batch run?

Accepted Answer

Coefficient of variation of bead MFI values across all acquisition tubes

Answer

Tracking bead MFI CV across tubes monitors instrument stability, laser fluctuation, and alignment consistency throughout a multi-tube acquisition session.

Question 3

When using viSNE (Cytobank implementation of t-SNE), which parameter controls the size of the neighborhood considered during embedding?

Accepted Answer

Perplexity

Answer

Perplexity in t-SNE/viSNE controls the effective number of neighbors considered, balancing attention between local and global data structure in the resulting 2D map.

Question 4

In phospho-flow cytometry data analysis, why is it important to use unstimulated cells as a reference rather than an FMO control?

Accepted Answer

The biological baseline of phosphorylation (unstimulated state) defines the true negative population for stimulation comparisons

Answer

In phospho-flow, the relevant comparison is between stimulated and unstimulated cells; the unstimulated sample defines the basal phosphorylation level that serves as the functional negative reference.

Question 5

What is the purpose of 'back-gating' in hierarchical flow cytometry analysis?

Accepted Answer

Applying a gate defined in a later analysis step to an earlier scatter plot to verify gate placement

Answer

Back-gating overlays a population identified in a later gating step onto an earlier scatter plot (e.g., FSC vs. SSC), confirming that the population is appropriately positioned and not contaminated by debris.

(SCYM) Specialist in Cytometry Practice Test

(SCYM) Specialist in Cytometry Practice Test

SCYM Data Analysis 5