Cytation 5 cell imaging multi mode reader

Collected blood samples were directly put into STRECK tubes and processed within 72 h. CTC enrichment utilised CTChip®FR1 biochip analysis performed on a ClearCell®FX1 System (Biolidics, Singapore) after lysis of red blood cells in 7.5 ml blood samples and centrifugation at 500 g at room temperature for 10 minutes, followed by resuspension of cell pellets.^{15 (link),16 (link),20 (link)} The output CTC enriched sample was loaded and fixed on a poly-l-lysine slide for subsequent enumeration of CTCs by immunofluorescence (IF) staining according to standard protocols. CTC isolation and enumeration were performed with pan-keratin (CK), which recognises keratins 4, 5, 6, 8, 10, 13 and 18. Cells were pre-hybridised with blocking solution and then hybridised to a cocktail of antibodies of pan-CK/EpCAM (Cell Signaling Technology, USA) and CD45 (BD Biosciences, USA) conjugated with Alexa-555 and APC secondary antibodies and stained with DAPI, as previously described. The cell images obtained from metastatic NPC sample analysis were captured on a fluorescence scanner, the Cytation 5 Cell Imaging Multi-Mode Reader (BioTeck, USA). The imaged CTCs were counted by imaging software using CellProfiler and CellProfiler Analyst, as previously described.^{16 (link)} CTCs were defined as nucleated cells positive for DAPI and CK/EpCAM IF staining, but negative for CD45 IF staining.