SCYM Fluorochrome Selection 4 — Questions and Answers
Question 1: In spectral flow cytometry, what enables the use of more fluorochromes than detectors in conventional cytometry?
- Increased laser power
- Full spectral unmixing using reference spectra (Correct answer)
- Use of quantum dots exclusively
- Higher quantum yield dyes
Correct 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.
Question 2: Which characteristic makes quantum dots (Qdots) particularly advantageous for multicolor flow cytometry panels?
- They all share a single emission peak
- They are excited by a single UV source but emit at multiple tunable wavelengths (Correct answer)
- They have lower brightness than organic dyes
- They are not affected by photobleaching but cannot be used with blue lasers
Correct 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.
Question 3: Which fluorochrome should be avoided when performing phosphorylation assays that require methanol-based fixation?
- BV421
- FITC
- PE (Correct answer)
- APC
Correct 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.
Question 4: The relative brightness index (RBI) of a fluorochrome is calculated using which two parameters?
- Quantum yield and molecular weight
- Extinction coefficient and quantum yield (Correct answer)
- Stokes shift and emission peak wavelength
- Laser power and detector efficiency
Correct answer: Extinction coefficient and quantum yield
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?
- Assign all bright dyes to the blue 488 nm laser
- Distribute markers across all three lasers to balance detector usage (Correct answer)
- Use only red-laser dyes to minimize spillover
- Place all viability dyes on the violet laser only
Correct 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.
Question 6: Which viability dye is excluded from live cells and enters dead cells with compromised membranes, making it suitable for use with fixed samples?
- Annexin V-FITC
- 7-Aminoactinomycin D (7-AAD)
- LIVE/DEAD Fixable Aqua (Correct answer)
- Propidium Iodide (PI)
Correct 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.
Question 7: Which laser is required to excite the commonly used fluorochrome PerCP-Cy5.5?
- 405 nm violet laser
- 638 nm red laser
- 488 nm blue laser (Correct answer)
- 355 nm UV laser
Correct 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.
SCYM Fluorochrome Selection 4
In spectral flow cytometry, what enables the use of more fluorochromes than detectors in conventional cytometry?