SCYM Fluorochrome Selection 4

Question 1

In spectral flow cytometry, what enables the use of more fluorochromes than detectors in conventional cytometry?

Accepted Answer

Full spectral unmixing using reference spectra

Answer

Spectral flow cytometry captures the full emission spectrum of each cell and uses unmixing algorithms with reference spectra to resolve overlapping fluorochromes.

Question 2

Which characteristic makes quantum dots (Qdots) particularly advantageous for multicolor flow cytometry panels?

Accepted Answer

They are excited by a single UV source but emit at multiple tunable wavelengths

Answer

Quantum dots can all be excited by a single UV or violet laser while emitting at different, size-tunable wavelengths, simplifying laser requirements.

Question 3

Which fluorochrome should be avoided when performing phosphorylation assays that require methanol-based fixation?

Accepted Answer

PE

Answer

PE is sensitive to methanol and can be destroyed or spectrally altered by methanol-based permeabilization, making it unsuitable for phospho-flow assays.

Question 4

The relative brightness index (RBI) of a fluorochrome is calculated using which two parameters?

Accepted Answer

Extinction coefficient and quantum yield

Answer

RBI is the product of a fluorochrome's molar extinction coefficient and quantum yield, predicting its theoretical brightness.

Question 5

When building a 10-color panel on a 3-laser instrument (405/488/638 nm), which strategy best distributes fluorochromes?

Accepted Answer

Distribute markers across all three lasers to balance detector usage

Answer

Distributing fluorochromes across all available lasers balances detector usage and reduces intra-laser spectral overlap.

(SCYM) Specialist in Cytometry Practice Test

(SCYM) Specialist in Cytometry Practice Test

SCYM Fluorochrome Selection 4