Facsymphony a1 cell analyzer

High-binding ELISA microplates were precoated with GNL prior to the immobilization of 5 μg/mL GPC-I53-dn5B, incubated at 4°C overnight, washed with 1X Tris-Buffered Saline, and blocked with 1% BSA in PBS. PBMC were obtained from leukapheresis products of healthy donors by Ficoll-Paque density gradient following the manufacturer’s protocol. NK cells were enriched by positive selection from human PBMCs using Miltenyi’s CD56 MicroBeads following manufacturer’s protocol and stimulated with 10 ng/mL IL-15 in Iscove's Modified Dulbecco's Medium supplemented with 10% FCS and 100 U/mL penicillin/streptomycin. Stimulated NK cells were incubated at 37°C overnight. On the day of the experiment, a 1:50 dilution of guinea pig serum in 1% BSA in PBS was added onto the well and incubated for 1 h at 37°C. NK cells were added onto the plate at 35,000 cells per well and incubated for 3 h at 37°C. After incubation, NK cells were transferred into a 96 well V-bottom microplate and stained with anti-CD16-PE (Biolegend) at a 1:1000 dilution (CD16 is a marker for NK cell activation⁶² (link)). As a positive control, phorbol myristate acetate (PMA) (50 ng/mL) and ionomycin (500 ng/mL) was added, and no serum wells was used as a negative control. Samples were read using a BD FACSymphony A1 Cell Analyzer.

HBEC3KT cells were removed from cell culture dishes using Accutase (ThermoFisher Scientific) and resuspended in 100μl 1×PBS. For viability, cells were incubated at room temperature for 10 min with 0.2 μl Zombie Aqua Fixable Viability stain (BioLegend) in 100 μl PBS. Control PE-mIgG2a, κ isotype (BioLegend, 400213), or PE-IL-28RA antibody (BioLegend, 337804) was added and incubated on ice for an additional 20 min. After incubation, the cells were washed with 1× PBS + 2% FBS and data were collected on a BD FACSymphony A1 Cell Analyzer (BD Biosciences) and analysis was performed using FlowJo software (FlowJo).

For cycle analysis, LNCaP WT and LNCaP MM were harvested, collected and washed in ice-cold PBS. Cells were fixed in ice-cold EtOH 70% at 4 °C for 1 h, then washed in PBS. Afterward, cell pellet was incubated in 100 μl PBS containing 0.5 μg/ml RNaseA (Life Tech) for 30 min at 37 °C. Cells were incubated with 100 μL propidium iodide (50 μg/mL, Life Tech) in PBS for 30 min at room temperature before the analysis. Cell death was determined with Annexin-V-FITC and propidium iodide staining according to manufacturer’s instructions (Annexin V FITC Kit, Miltenyi Biotec). For FACS analysis a BD FACSymphony™ A1 Cell Analyzer (BD Biosciences) was used, and data were analyzed and quantified with FlowJo software (Treestar). FACS analysis were performed in three independent biological replicates; representative data are shown.