Anti ifn γ apc antibody

Healthy human PBMCs were seeded at 0.5 million cells/ml in RPMI (10% FBS, 1% Pen/Strep) in a 24-well plate. TLR agonists were incubated with the PBMCs overnight at 1 μM. The supernatant was collected and analyzed for cytokine concentrations. Target cells were trypsinized and added to the wells at an effector to target ratio of 2:1. Cetuximab was added at a final concentration of 200 nM. Anti-CD107a APC Cy7 (Biolegend, San Diego, CA) was added to all samples. The samples were incubated at 37 °C. One hour later, Brefeldin A (Biolegend, San Diego, CA) was added and the samples were once again incubated at 37 °C. Four hours later, cells were collected and stained for extracellular markers using anti-CD3 FITC, anti-CD56 Brilliant Violet 650, anti-CD4 PE-Cy7, anti-CD8 Brilliant Violet 605, and anti-CD69 PerCP-Cy 5.5 (Biolegend, San Diego, CA) antibodies. Intracellular staining was performed with anti-IFN-γ APC antibody (Biolegend, San Diego, CA) using eBioscience Foxp3 Transcription Factor Staining Buffer Set (Thermofisher, Waltham, MA). All samples were set up in duplicates. Samples were analyzed using a BD Fortessa H0081 flow cytometer at the University of Minnesota Flow Cytometry Core Facility.

PBMCs were seeded (5 × 10⁵ cells/well) in 96-well
U-bottom plates and treated with the compounds (1 μM) or vehicle
(0.1% DMSO) for 24 h. Anti-CD107a FITC and monensin (Biolegend, San
Diego, CA) were added to all wells for the last 4 h of incubation.
The cells were washed and stained with Live/Dead Fixable Aqua Dead
Cell Stain (Invitrogen, Carlsbad, CA). After further washing, Fc receptors
were blocked with Human TruStain FcX (Biolegend) and cells were stained
for extracellular markers using anti-CD3 APC/Fire 750, anti-CD56 PE,
and anti-CD69 PerCP-Cy5.5 antibodies (Biolegend). Intracellular staining
was performed with an anti-IFN-γ APC antibody (Biolegend) after
fixation and permeabilization using the Cyto-Fast Fix/Perm Buffer
Set (Biolegend). Samples were analyzed using an Attune NxT flow cytometer
(Thermo Fisher Scientific, Waltham, MA) and the FlowJo software (Tree
Star, Inc., Ashland, OR). Following exclusion of dead cells, CD3^- CD56⁺ NK cells were evaluated for expression
of CD107a, CD69, and IFN-γ. The gating strategy is described
in detail in Figure S6. Statistical significance
was determined with one-way ANOVA with subsequent Bonferroni’s
multiple comparisons test.