Sputum processing
Sputum was dissociated and labeled using recently published protocols [A small aliquot of cells was set aside for use as controls while the majority was divided into two tubes for the main analysis. Both tubes were labeled with Fixable Viability Stain 510 (FVS510) and CD45-PE. One tube, the “blood tube”, received CD66b-FITC (to identify granulocytes), CD3-Alexa-Fluor-488 (T-cells), CD19-Alexa-Fluor-488 (B-cells) and CD206-PE-CF594 (macrophages). In the other tube, the “epithelial tube”, cells were labeled with the epithelial cell markers pan-cytokeratin (Pan-CK)-Alexa-Fluor-488 and EpCAM-PE-CF594. Cells were incubated for 35 min on ice, protected from light. After washing with HBSS, cells were fixed and stored on ice until the next day, when a TCPP solution (20 µg/mL) was added (3.3 × 10