ISAC Analytical Methods and Techniques 3

Question 1

What is the purpose of an FMO (Fluorescence Minus One) control in multicolor flow cytometry?

Accepted Answer

To define gate boundaries accounting for spectral spillover from all other channels

Answer

FMO controls contain all fluorochromes except the one being gated, revealing the true spread of spillover signal into that channel to set accurate gate boundaries.

Question 2

During cell sorting, what is the function of the piezoelectric crystal in the sort stream?

Accepted Answer

It oscillates to break the fluid stream into uniform droplets

Answer

The piezoelectric crystal vibrates at a defined frequency to break the fluid stream into uniformly sized droplets at a precise break-off point.

Question 3

Which gating strategy best reduces doublet contamination in cell cycle analysis?

Accepted Answer

Gating on FSC-A vs FSC-H or FSC-W

Answer

Plotting FSC-A (area) vs FSC-H (height) exploits the fact that doublets produce disproportionately high area signals, enabling their exclusion.

Question 4

In intracellular cytokine staining (ICS), why is brefeldin A or monensin used?

Accepted Answer

To block protein secretion and retain cytokines inside cells

Answer

Brefeldin A and monensin block Golgi-mediated protein secretion, causing cytokines to accumulate intracellularly where they can be detected by flow cytometry.

Question 5

What statistical measure is most appropriate for describing the fluorescence intensity distribution of a heterogeneous cell population?

Accepted Answer

Geometric mean fluorescence intensity

Answer

Geometric mean is preferred for log-scale fluorescence data because it accurately represents the central tendency of log-normal distributions without bias from extreme values.

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

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

ISAC Analytical Methods and Techniques 3