A method for high purity sorting of rare cell subsets applied to TDC

TDC are a recently described subset of polyclonal αβ T-cells with dendritic cell properties. Because of their low number in peripheral immune compartments, isolation and characterization of TDC with existing purification methods are technically challenging. Here we describe a customized gating strategy and a flow cytometry-based cell sorting protocol for isolation of TDC. The protocol was developed because, despite very conservative gating for dead-cell and doublet exclusion, cells obtained with normal sorting procedures were enriched for TDC but not pure. Re-sorting the output of the first round of sorting results in highly pure TDC. Cells obtained with this method are viable and can be used for in vitro characterization. Moreover, this double-round sorting strategy can be universally applied to the isolation of other rare cell subsets.