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

Recovery is a key performance parameter in cell sorters, a metric that assesses the match between the number of particles reported as sorted by the instrument and the actual number of particles gathered in the collection vessels. Sorting relies on the precise timing of the charging of a droplet containing the particle of interest in a critical measurement called drop delay (DD). DD timings are typically reliant on manufacturer recommended fluorescent bead reagents. Cuvette-based cell sorters in particular depend upon these fixed-sized QC beads for an automated approach to the DD calculation using an image-based camera system. Previous literature has highlighted the mismatch between these DD values and the settings best accommodating actual samples. Here, we present a new method for DD calculation-the Three-Puddle Method (3PM), based on procedures originally described for jet-in-air cell sorters; it optimizes DD values according to the target particle to be sorted, increasing sort recoveries for a range of cell sizes and particle types. With regard to recovery, 3PM-calculated DD values correlate with those achieved via optimum DD, defined using Rmax protocol, a robust metric for recovery. The advantages of the 3PM then are that it is a simple-to-implement protocol which has limited cell expenditure, essential in the handling of precious rare samples and the success of single cell and bulk sorting and the downstream applications relying on it.