Fluorochrome Selection Flashcards

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

Answer: Full spectral unmixing using reference spectra

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

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

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

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

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

Answer: PE

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

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

Answer: Extinction coefficient and quantum yield

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

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

Answer: Distribute markers across all three lasers to balance detector usage

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

Which viability dye is excluded from live cells and enters dead cells with compromised membranes, making it suitable for use with fixed samples?

Answer: LIVE/DEAD Fixable Aqua

LIVE/DEAD Fixable dyes react covalently with cellular amines, allowing dead-cell discrimination even after fixation, unlike PI or 7-AAD.

Which laser is required to excite the commonly used fluorochrome PerCP-Cy5.5?

Answer: 488 nm blue laser

PerCP-Cy5.5 is excited by the 488 nm blue laser and emits around 695 nm, making it standard on blue-laser configurations.