Coculture and multi-parametric flow cytometry
Cryopreserved PBMCs were thawed and resuspended in RPMI overnight in the
presence of 120 IU/mL (Peprotech) before coculture. C17 and NPC43+ve NPC
cell lines were pre-treated with indicated doses of IDX and/or cis for
24 h before coculturing with PBMCs via tranwell (3 µm pore-size,
Corning) for 3 days. For surface staining, PBMCs were collected, washed
and resuspended in FACS buffer (PBS, 0.5 % BSA and 2 mM EDTA). Cells
were pretreated with Fc block reagents (1:200; Biolegend), then stained
with a mix of surface fluorophore-conjugated anti-mouse antibodies
listed in Supplementary Table 1. For intracellular Ki67 staining, PBMCs
were stimulated with anti-CD3/CD28 antibodies (BD Biosciences, both at
1:1000 dilution) and cocultured with tumor cells for 5 days. Brefeldin A
(1 µg/mL; Sigma-Aldrich) was added during the last 5 h of culture. After
coculture, cells were washed, surface stained (4 °C for 30 min) and then
fixed with Cytofix/Cytoperm, followed by intracellular Ki67 staining.
After the staining, cells were washed again prior to processing on a
NovoCyte Quanteon flow cytometer (Acea Biosciences). Data analysis was
perfoemed using FlowJo software (FlowJo, LLC, USA). Nonviable cells were
excluded from the analysis based on forward and side scatter profiles,
and 7-AAD dead cell stain.