Ifn g xmg1

Antibodies for in vivo applications (CD4 (YTS191.1.2), CD8 (YTS169.4.2), NK1.1 (PK136), IFN-g (R4-6A2), Il-1b (BE0246) and isotype controls were produced by our in-house facility or purchased from BioXcell. For flow cytometry, single-cell suspensions of spleen were prepared according to standard protocols. Flow cytometric analysis were performed by using anti-mouse mouse CD4 (GK1.5), CD3 (145-2C11), CD8b (53-6.7), CD62L (MEL-14), CD127 (A7R34), KLRG1 (2F1), NK1.1 (PK136), CD11b (M1/70), CD11c (N418), CD86 (GL1), GR1 (RB6 8C5), F4/80 (BM8), TNF (MP6-XT22), IFN-g (XMG1.2) and granzyme B (NGZB) from eBioscience, preceded by blocking of Fc receptors using 2.4G2 antibodies (in house generated). To measure cytokine production, cells were stimulated with 1 mg of MCMV peptides M57 (SCLEFWQRV) or m139 (TVYGFCLL) or with SIINFEKL (N4) peptide for 4 h in the presence of Brefeldin A (10 mg/ml; eBioscience). MHC class I tetramers were provided by A. ten Brinke (Amsterdam, the Netherlands). For intracellular staining, permeabilization and fixation of cells was done with the Fix/Perm kit (BD Biosciences). All data were acquired using a FACSVerse (BD Biosciences) and analyzed using FlowJo software (Tree Star).