ISAC Panel Design and Fluorochrome Selection

Question 1

What is the primary consideration when selecting fluorochromes for a multicolor flow cytometry panel?

Accepted Answer

Spectral overlap between fluorochromes

Answer

Spectral overlap between fluorochromes is the primary concern because emission bleed-through into neighboring channels requires compensation, which can degrade resolution.

Question 2

In panel design, a bright fluorochrome like PE should ideally be assigned to which type of marker?

Accepted Answer

Dimly expressed or rare markers

Answer

Assigning bright fluorochromes like PE to dimly expressed antigens maximizes signal from those targets and improves their detection above background.

Question 3

What is spectral spillover in flow cytometry?

Accepted Answer

Detection of one fluorochrome's emission in another fluorochrome's detection channel

Answer

Spectral spillover occurs when a fluorochrome's emission spectrum extends beyond its primary detection channel into channels designated for other fluorochromes.

Question 4

Which parameter is most important for evaluating fluorochrome brightness in the context of panel design?

Accepted Answer

Staining index

Answer

The staining index quantifies the separation between positive and negative populations relative to background spread, making it the most practical measure of effective brightness for panel decisions.

Question 5

What is a tandem dye in flow cytometry?

Accepted Answer

A fluorochrome consisting of two energy-coupled dyes that shift emission to a longer wavelength

Answer

Tandem dyes couple a donor fluorochrome to an acceptor via FRET, so energy absorbed by the donor is transferred and emitted at the acceptor's longer wavelength.

(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