Excluding Doublets in FACS Analysis: A Comprehensive Guide

• FSC (Forward Scatter):Measures cell size by detecting scattered light in the forward direction.
• SSC (Side Scatter): Measures cell granularity or internal complexity based on side-scattered
  light.

While FSC and SSC alone may not always distinguish doublets, they are essential for primary cell gating.

• Area (A): Represents the total integrated signal under the pulse and is directly proportional to cell size.
• Height (H): Measures the peak of the signal pulse; a doublet may have a higher peak than a single cell.
• Width (W): Reflects the pulse duration as the cell passes through the laser beam. Doublets tend to produce wider pulses than single cells.

 Plot FSC-A vs. FSC-H for Doublet Identification

• Rationale Doublets generate a higher peak height relative to area compared to single cells.
• Procedure: Plot FSC-A (area) on the x-axis and FSC-H (height) on the y-axis.
• Expected Pattern: Single cells form a tight, linear population along a diagonal, as area and height correlate directly for single cells. Doublets and larger aggregates deviate from this line.
• Gating Strategy: Gate around the linear population to retain single cells and exclude
  events above or below this line.

Plot FSC-W vs. FSC-H for Enhanced Doublet Discrimination

• Rationale: Doublets produce wider pulses due to the combined width of two cells passing together.
• Procedure: Plot FSC-W (width) on the x-axis and FSC-H (height) on the y-axis.
• Expected Pattern: Single cells will have a narrow width and a tight cluster, while doublets will appear as a distinct population with higher width.
• Gating Strategy: Exclude events with higher FSC-W, which are likely doublets, by gating around the single-cell population.

• Rationale: SSC can also help exclude doublets, particularly in heterogeneous samples.
• Procedure: Plot SSC-A vs. SSC-H or SSC-W vs. SSC-H to examine signal characteristics in side scatter. 
• Expected Pattern: Like FSC, single cells will form a tight population, while doublets will exhibit a broader SSC-W or deviate from the SSC-A vs. SSC-H linearity.
• Gating Strategy: Use SSC parameters if additional discrimination is needed, particularly in
  samples with granularity differences among cell types.

For samples with fluorescent markers, fluorescence parameters can provide additional doublet discrimination.

• Rationale: Doublets display higher fluorescence due to two cells with combined fluorescence signals, which can increase both area and height disproportionately.
• Procedure: Plot Fluorescence Area (Fl-A) vs. Fluorescence Height (Fl-H) for each fluorescent channel.
• Expected Pattern: Single cells form a diagonal pattern where area and height correlate linearly. Doublets and aggregates deviate from this line due to increased fluorescence.
• Gating Strategy: Gate along the linear population to exclude fluorescence events with disproportionate height or area, indicative of doublets.

1. Gate FSC-A vs. FSC-H: Retain events along the linear line to exclude primary doublets.
2. Gate FSC-W vs. FSC-H: Exclude events with higher FSC-W, which are likely doublets or larger
   aggregates.
3. Apply SSC Parameters (if needed): For more complex samples, use SSC-A vs. SSC-H or SSC-W vs. SSC-H to refine doublet exclusion further.
4. Use Fluorescence Gating (Fl-A vs. Fl-H): For fluorescently labeled cells, add a fluorescence-based check to ensure high-fluorescence doublets are excluded.

• Run control samples: Use a sample of known single cells to validate your gating strategy.
• Optimize gating for cell type: Different cell types or experimental conditions may require adjustments in gating parameters.
• Verify with multiple parameters: Using combinations of FSC, SSC, and fluorescence plots enhances accuracy in doublet exclusion.

By following a systematic approach to doublet exclusion, you can ensure more accurate FACS data analysis, leading to reliable and interpretable results across various applications.