Panel Design and Fluorochrome Selection Flashcards

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

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

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

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

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

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.

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

Answer: Epitope competition that reduces binding efficiency and signal from both antibodies

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.

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

Answer: Membrane-impermeant dyes such as propidium iodide or DAPI

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.

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

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

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.

Which combination of controls is recommended when validating a new multicolor flow cytometry panel?

Answer: Unstained cells, single-color controls, FMO controls, and fully stained samples

A complete validation set includes unstained cells for autofluorescence baseline, single-color controls for compensation, FMO controls for accurate gate placement, and fully stained samples to assess overall panel performance.

In flow cytometry data analysis, what is 'backgating' used for?

Answer: Returning to a previous bivariate plot to verify that a gated population displays expected marker expression

Backgating involves plotting a gated population on earlier plots to confirm it expresses the markers expected for that population, serving as a quality check on the gating strategy.