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Efficient sorting methods are required for the isolation of cellular subpopulations in basic science and translational applications. Despite this, throughputs, yields, viabilities, and processing times of common sorting methods like fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) are underreported. In the current study, we set out to quantify the ability of these sorting methods to separate defined mixtures of alkaline phosphatase liver/bone/kidney (ALPL)-expressing and non-expressing cell types. Results showed that initial MACS runs performed using manufacturer’s recommended antibody and microbead concentrations produced inaccurate ALPL+ vs. ALPL− cell splits compared to FACS when ALPL+ cells were present in larger proportions (>~25%). Accuracy at all proportions could be achieved by using substantially higher concentrations of labeling reagents. Importantly, MACS sorts resulted in only 7–9% cell loss compared to ~70% cell loss for FACS. Additionally, MACS processing was 4–6 times faster than FACS for single, low proportion samples but took similar time for single, high-proportion samples. When processing multiple samples, MACS was always faster overall due to its ability to run samples in parallel. Average cell viability for all groups remained high (>83%), regardless of sorting method. Despite requiring substantial optimization, the ability of MACS to isolate increased cell numbers in less time than FACS may prove valuable in both basic science and translational, cell-based applications.

Considerations for high-yield, high-throughput cell enrichment: fluorescence versus magnetic sorting