Compensation Techniques Flashcards
7 cards from real SCYM practice questions. Tap to flip, then mark Knew It or Still Learning — missed cards come back until you master them.
Read the first 7 Compensation Techniques flashcards as text
What is the primary reason tandem dye degradation poses a special challenge for compensation?
Answer: Degraded tandems emit at the donor wavelength, altering spillover into neighboring channels
Tandem dye breakdown causes incomplete energy transfer, releasing donor emission and increasing spillover into donor-wavelength detectors.
When should compensation be set AFTER gating rather than before, in an analysis workflow?
Answer: Never — compensation must be applied before any gating
Compensation is always applied to raw data before gating because it corrects signal values that gates depend on.
Which statistical measure is recommended for setting compensation to minimize spreading error in dim populations?
Answer: Median fluorescence intensity (MFI)
MFI (median) is preferred because it is robust to outliers and skewed distributions caused by spreading error in dim populations.
A researcher notices that after compensation, a double-negative population forms a 'bowtie' shape on a two-parameter dot plot. What does this indicate?
Answer: Over-compensation in at least one channel
A bowtie or butterfly shape indicates over-compensation, where high-signal events are pulled below the negative population mean.
What is the purpose of including an 'unstained' or 'negative' control in compensation workflows?
Answer: To define the baseline autofluorescence for determining true negative populations
The unstained control defines autofluorescence background, enabling accurate identification of true negative cells separate from compensated positives.
In a panel where Alexa Fluor 700 and APC-Cy7 are both used, which compensation challenge is MOST anticipated?
Answer: High spillover from APC-Cy7 into the Alexa Fluor 700 detector
APC-Cy7 has significant emission that spills into the Alexa Fluor 700 detection window because both are excited at 633/647 nm and detected in adjacent red channels.
Which approach best mitigates compensation error introduced by cell-specific autofluorescence differences between control beads and experimental cells?
Answer: Using cell-based single-color controls from the same cell type as the experiment
Cell-based controls inherently carry the same autofluorescence as experimental cells, making them the most accurate reference for compensation.