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.