Abstract
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.
Highlights
Efficient sorting methods are required for the isolation of cellular subpopulations in basic science and translational applications
Manufacturer’s protocol magnetic-activated cell sorting (MACS) runs displayed major differences in retained and flowthrough output fractions compared to fluorescence-activated cell sorting (FACS)-separated alkaline phosphatase liver/bone/kidney (ALPL)+ and ALPL− fractions (Fig. 1)
To determine whether antibody and microbead concentrations specified by the manufacturer’s MACS protocol was responsible for the observed differences, MACS antibody- and microbead- treatment concentrations were varied in a series of optimization trials to determine their effect on isolation of fixed, ALPL+stromal vascular fraction (SVF) cells
Summary
Efficient sorting methods are required for the isolation of cellular subpopulations in basic science and translational applications. It lacks the sensitivity and cell-specific data provided by a fluorescence-based system and is not compatible with multiple-marker profiles. A general ASC definition proposed by the International Federation of Adipose Therapeutics and Science (IFATS) includes positive/negative expression for four surface markers (CD34+/CD31−/CD45−/CD235a−), with an additional four markers for increased specificity (CD13, CD73, CD90, and CD105)[15] These restrictive definitions result in very small numbers of enriched, yet still heterogeneous, cells such that the population input to FACS must be extremely large to acquire therapeutically relevant numbers (~106–108) as output[16,17,18,19,20,21,22]. Groups have isolated subpopulations of induced pluripotent stem cells and jaw periosteal cells based on ALPL expression that were capable of increased osteogenesis, though this has not yet been demonstrated with primary SVF cells[29,30]
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