Automated flow cytometric selection of viable single cells
Heuristics-guided
EVENT, LUNG, EVENTS, BSE
The first stage of the CyPath Lung assay is the automated FCM identification of viable single cells (Fig. Automated gating of FCM data. Each sample run included polystyrene beads of known diameter (5–30 µm NIST beads), compensation tubes for each fluorochrome channel used, unstained sputum, and an antibody isotype sputum control. Each tube corresponded to a single Flow Cytometry Standard (FCS) file that contained sample metadata and per event values for each light and fluorescence channel acquired plus a Time parameter recorded as tubes are run.As shown in Fig. Events within the bead size exclusion (BSE) gate were then restricted to exclude a population with unusual FSC and SSC height profiles (Fig. Cells below the threshold for FVS510-positivity were retained (Fig. Heuristics-guided Viability Gate Setting. A “singlets” gate (FSC-Area vs FSC-Width) excluded cell doublets or small aggregates (Fig. Heuristics-guided Singlets Gating. 