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

FMO controls contain every fluorochrome except the one being gated, so the signal seen in that channel represents only the spread from other fluorochromes, enabling accurate gate placement.

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

Spreading error is the additional variance introduced when compensation is applied to correct for spillover; it increases noise and can reduce the ability to resolve closely spaced populations.

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

Choosing fluorochromes whose emission peaks are widely spaced minimizes spectral overlap, allowing more independent detection channels to be used per laser line.

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

PE's very high quantum yield combined with a large absorption cross-section makes it one of the brightest fluorochromes available, ideal for detecting low-abundance targets.

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

Tandem dyes are susceptible to dye degradation and lot-to-lot spectral variability because partial breakdown of the FRET pair causes donor emission bleed-through, complicating compensation.

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

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

ISAC Panel Design and Fluorochrome Selection 2