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

The fluorochrome brightness alone

Answer

The manufacturer's geographic location

Answer

Whether the clone has been used in publications from the same laboratory

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

A dedicated channel for measuring instrument noise and background

Answer

The detection channel with the lowest signal-to-noise ratio in a panel

Answer

A fluorochrome channel reserved for doublet discrimination

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

Fluorochrome color interference between the two labels

Answer

Increased total reagent cost per sample

Answer

Signal amplification artifacts on the detector

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

FITC-conjugated isotype control antibody

Answer

PE-conjugated lineage marker antibody

Answer

APC-conjugated secondary antibody

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

It determines the physical aperture size of the flow cell

Answer

It controls the sample volumetric flow rate through the instrument

Answer

It affects only the side-scatter signal, not fluorescence

Question 6

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

Accepted Answer

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

Answer

Single-color controls only, to minimize reagent use

Answer

Fully stained samples and isotype controls only

Answer

Predicted optimal settings generated by panel design software alone

Question 7

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

Accepted Answer

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

Answer

Increasing laser power retroactively to improve dim signal detection

Answer

Removing low-scatter debris events from all plots simultaneously

Answer

Recalibrating photomultiplier tube voltages after acquisition