Pe anti mouse h 2kb h 2db

For staining of MHC-I, cells were treated for 24 hrs with the indicated concentration of IFNγ and one million cells were resuspended in PBS supplemented with 0.5% BSA and 2mM EDTA (PBE). Cells were incubated for 5 min with 1ug/ml of anti-CD16/32 (2.4G2, BioXcell) at room temperature. Cells were washed with PBS and stained with Zombie fixable viability dye (Biolegend) 15 min at room temperature. Cells were washed with PBE and stained with PE anti-mouse H-2Kb/H-2Db or PE anti-human HLA-A,B,C (Biolegend) antibody for 20 min at 4°C. Cells were washed and resuspended in PBE. Samples were acquired on the BD FACSymphony. Data were analyzed using FlowJo v.10.0.8 software.
For staining of TNFRI, one million cells were resuspended in PBS supplemented with 1% BSA and 1mM EDTA (FACS buffer). Cells were washed with FACS buffer and stained with APC anti-mouse TNFRI (Biolegend) antibody for 15 min at 4°C in the dark. Cells were washed and resuspended in FACS buffer with 60 ng/mL DAPI. Samples were acquired on the BD FACSymphony. Data were analyzed using FlowJo v.10.0.8 software.

The following antibodies were used in flow cytometry: PE anti-mouse H-2K^b/H-2D^b, PE anti-mouse IFNγR β chain (Biolegend, San Diego, CA, USA), PE anti-mouse CD119 (IFNγ Receptor 1) (eBioscience, San Diego, CA, USA). Antibodies used in western blotting analysis were: iNOS, pTyr701-Stat1, total Stat1, total Stat2, pSer536-p65, total p65 pSer32-IκBα, total IκBα, β-actin, GAPDH (Cell Signaling Technology, Danvers, MA, USA); pTyr690-Stat2 (Abcam, Cambridge, MA, USA); LaminB (Epitomics, Burlingame, CA, USA). Recombinant mouse IFNα, IFNβ, IFNγ and TNFα were from R&D Systems (Minneapolis, MN, USA). L-NMMA was from Sigma-Aldrich (St. Louis, MO, USA).