A method of quantifying cell sorting yield in "real time"

Abstract

Cell sorting flow cytometers sort cells by applying electrical charges to a stream that forms liquid droplets containing the cells at a set time after sample interrogation. The correct time to apply these charges is determined based on calibration beads and automated technology. The central tenet of this method is that beads accurately indicate the yield of cells sorted using the same instrument parameters. HEK293T cells were incubated with Accudrop™ calibration beads. Cell incorporation of beads by phagocytosis was confirmed by imaging cytometry. Cells containing beads were analyzed and sorted on unmodified commercially available cell sorters that have automated technology for setting drop delay times. Based on cell sorter drop delay times optimized using beads, sorting experiments demonstrate that yield can be assessed in real time using bead loaded cells and existing technology. Here, data presented show that cells have lower sorting yields than indicated using beads. Further, this data show clear trends related to nozzle tip diameter and sorting yield. The present study demonstrates a method to quantify yield "on the fly" during cell sorting, that is separated from drop charge counts and removes the variables associated with yield assessment from collection tubes. These data have implications for the expected recovery of cells from sorting experiments.