Increasing Cell Sorting Recovery Using the Simple "Three-Puddle Method".

Area of Science:

• Biotechnology
• Cell Biology
• Analytical Chemistry

Background:

• Cell sorter recovery is critical, depending on precise drop delay (DD) timing.
• Current DD calculations often use manufacturer beads, leading to mismatches with actual sample sorting.
• Optimizing DD is essential for accurate cell sorting and downstream applications.

Purpose of the Study:

• To introduce and validate the Three-Puddle Method (3PM) for optimizing drop delay (DD) in cell sorters.
• To improve cell sorting recovery rates across diverse particle types and sizes.
• To provide a simple, low-cell-expenditure protocol for accurate DD calculation.

Main Methods:

• The Three-Puddle Method (3PM) was adapted from jet-in-air sorter procedures for cuvette-based systems.
• 3PM optimizes DD values based on the specific particle being sorted.
• Drop delay (DD) optimization was validated against the Rmax protocol, a recovery metric.

Main Results:

• 3PM-calculated DD values showed strong correlation with optimum DD settings determined by the Rmax protocol.
• The method enhances sort recovery for a range of cell sizes and particle types.
• 3PM offers a simple, low-cost alternative to traditional DD calibration methods.

Conclusions:

• The Three-Puddle Method (3PM) is an effective and accessible protocol for optimizing cell sorter drop delay (DD).
• 3PM improves sorting recovery and is particularly beneficial for rare or precious samples.
• This method supports more reliable single-cell and bulk sorting for downstream analyses.