Flow cytometry
Whole blood (100 μL) or 1 x 106 CIK infusion cells were stained for cell surface markers to analyze T cell differentiation status. The following antibodies were used: anti-CD45RA-FITC; anti-CCR7-PE, anti-CD3-PC7; anti-CD8-APC, anti-CD45RO-PC7 (Tube1); anti-CD45-APC, anti-CD25-PE, anti-Helios-FITC; anti-CD4-PC7; anti-FoxP3-PC5.5 (Tube2); anti-CD45-APC, anti-CD8-PE, anti-CD56-FITC; anti-CD4-PC7; anti-CD3-PC5.5 (Tube3); anti-CD45-APC, anti-CD8-PE, anti-HLA-DR-FITC; anti-CD4-PC7; anti-CD3-PC5.5 (Tube4); anti-CD45-APC, anti-CD16-PE, anti-CD1c-FITC; anti-CD11c-PC7. All antibodies were titrated before use, and fluorescence-minus-one (FMO) controls were created for each antibody panel to set gates for positive events. For anti-lineage-PC5.5 (Tube5), cells were washed with phosphate-buffered saline (PBS) and stained for viability using LIVE/DEAD Fixable Violet (Molecular Probes) for 15 min, washed once and resuspended in fluorescence-activated cell sorting (FACS) buffer consisting of PBS, 1% BSA and 5 mM EDTA. Cells were then incubated with the above indicated antibodies for 1 h at 4°C. Samples were washed three times with FACS buffer and fixed in 1% paraformaldehyde. Positively-stained cells were differentiated from background using FMO controls. Flow cytometry was performed using the BD LSR Fortessa flow cytometer. Analysis was performed using Flowjo software (Tree Star Inc. version 10.1). Absolute cell numbers were determined by relating the CD45+cell count from flow cytometry to the total leucocyte count.