The introduction of full spectral technology in flow cytometry has facilitated access to an increasing number of markers to define cell subsets with higher precision. Cell sorting has a unique advantage to combine high throughput single cell analysis and recovery of rare live single cells for further downstream multi-omics analysis. Many studies have focused on advancing high dimensional single cell analysis; however, strategies to maximize cell sorting recovery in the context of deep immunophenotyping remain poorly defined. In this study, we evaluated sort performance in a six-way simultaneous cell sort setup. We modified a protocol using counting beads to assess absolute count in different sort decision criteria or modes. We demonstrate that the number of events collected can vary as much as 20% from the values indicated by the sort counter and is dependent on sort mode. Using the absolute count assay, we confirmed optimal conditions for six-way sorting of diverse human peripheral blood cell subsets defined by a 35-color panel and delineated pitfalls that can ultimately lead to suboptimal yield. Together, these findings provide novel insights into optimization of sort performance for advanced sorting and introduce a new approach for refining strategies for the simultaneous isolation of complex or rare cell subsets.
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