ISAC Panel Design and Fluorochrome Selection 2

Question 1

What is the purpose of a fluorescence minus one (FMO) control in a multicolor flow cytometry panel?

Accepted Answer

To define accurate gate boundaries by capturing spillover spread from all other fluorochromes

Answer

To measure laser power stability over time

Answer

To calculate absolute cell counts from a known volume

Answer

To assess cell viability independently of staining

Question 2

Which of the following best describes 'spreading error' in multicolor flow cytometry?

Accepted Answer

Increased variance in fluorescence measurements caused by spectral spillover and its mathematical compensation

Answer

Physical dispersal of cells across the laser interrogation point

Answer

Uneven conjugation of fluorochromes to antibodies

Answer

Technical errors introduced during data file export

Question 3

When designing a panel on an instrument with limited laser lines, which strategy best maximizes the number of parameters measured?

Accepted Answer

Selecting fluorochromes with widely separated emission peaks on each laser

Answer

Using only the two brightest fluorochromes available

Answer

Using multiple antibodies labeled with the same fluorochrome

Answer

Increasing laser power to resolve overlapping signals

Question 4

What characteristic makes PE (phycoerythrin) one of the most valuable fluorochromes in flow cytometry panels?

Accepted Answer

Its extremely high quantum yield and molar absorptivity resulting in exceptional brightness

Answer

Its narrow emission spectrum that eliminates spillover

Answer

Its ability to be excited simultaneously by all laser lines

Answer

Its resistance to photobleaching during extended acquisitions

Question 5

Which of the following is a recognized disadvantage of using tandem dyes in flow cytometry experiments?

Accepted Answer

They show lot-to-lot variability and can degrade, producing incomplete energy transfer artifacts

Answer

They cannot be conjugated to antibodies

Answer

They require ultraviolet lasers not available on standard instruments

Answer

They are incompatible with intracellular staining protocols

Question 6

In spectral flow cytometry, what replaces traditional bandpass filters for signal detection?

Accepted Answer

Prisms or diffraction gratings that disperse light across an array of detectors

Answer

Higher-powered lasers that reduce spectral overlap

Answer

Digital compensation matrices applied before acquisition

Answer

Avalanche photodiodes replacing photomultiplier tubes

Question 7

What does 'unmixing' refer to in spectral flow cytometry data analysis?

Accepted Answer

A mathematical process that deconvolves overlapping spectra to quantify each fluorochrome's individual contribution

Answer

Physically separating cells into distinct collection tubes

Answer

Removing doublets and debris from the data file

Answer

Normalizing laser power across acquisition runs