Gating strategy in flow cytometry to purify HCLc tumour cells and matched germline T cells.

<p>Splenocytes were stained with anti-CD19-eFluor450, anti-CD3-PE, anti-CD11c-APC, anti-CD27-APC-eFluor780 and anti-CD103-FITC prior to sorting on the FACSAria. The plots show sorting on a representative HCLc case. <b>A:</b> displays pre-sorted mononuclear cells, with CD19⁺CD11c⁺CD103⁺CD27^- HCLc tumour cells 78.0% of sample and CD3+ T cells 3.3%. Cells were initially gated on FSC/SSC (<b>A</b>, left) then either CD3+ for T cells or CD19+ for B cells (<b>A</b>, second left). CD19+ cells were subsequently gated as CD11c+ (<b>A</b>, second right), then CD103+ and CD27- (<b>A</b>, right) to capture high purity HCL cells. In <b>B</b> post-sorted cells were checked for purity: T cells were purified at 97.3% (left panel) and HCLc cells purified at 98.2% (right, three panels). Data is representative of cell fraction isolation in each of 5 samples.