ISAC Panel Design and Fluorochrome Selection 3

Question 1

Which factor is most critical when selecting antibody clones for inclusion in a multicolor flow cytometry panel?

Accepted Answer

Clone specificity, affinity, and potential cross-reactivity with non-target antigens

Answer

Clone specificity and affinity determine signal accuracy, while cross-reactivity with unintended antigens can produce false-positive results that confound panel interpretation.

Question 2

What is the 'dump channel' strategy in flow cytometry panel design?

Accepted Answer

Using one fluorochrome to simultaneously label multiple unwanted cell populations for exclusion in a single gate

Answer

The dump channel strategy labels all non-target cells (e.g., dead cells, debris, off-lineage populations) with a single fluorochrome so they can be excluded together, simplifying the gating hierarchy.

Question 3

When two antibodies from different clones target the same antigen in a panel, what is the primary concern?

Accepted Answer

Epitope competition that reduces binding efficiency and signal from both antibodies

Answer

Two antibodies binding the same or overlapping epitopes compete for a limited number of binding sites, reducing signal from each antibody and potentially causing underestimation of antigen expression.

Question 4

Which of the following reagents is most commonly used for live/dead cell discrimination in flow cytometry?

Accepted Answer

Membrane-impermeant dyes such as propidium iodide or DAPI

Answer

Propidium iodide and DAPI are excluded by intact cell membranes but enter and stain nucleic acids in dead cells with compromised membranes, enabling reliable viability discrimination.

Question 5

Why is a fluorochrome's excitation efficiency at the instrument's laser wavelength important in panel design?

Accepted Answer

It governs how many photons are absorbed and emitted, directly affecting brightness and signal-to-noise ratio

Answer

A fluorochrome's molar absorptivity at the specific laser wavelength determines how efficiently it is excited; poor excitation efficiency results in a dim signal even for a high-quantum-yield fluorochrome.

(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 3