Antimouse cd8 fitc clone kt15

The frequency of NS3-specific CD8+ T cells was analysed by ex vivo staining of splenocytes using the recombinant soluble dimeric mouse H-2D(b):Ig fusion protein (BD Biosciences, San Jose, California, USA) as described previously.21 29 (link) In brief, 1×10⁶ spleen cells were resuspended in PBS/1% FBS (FACS buffer) and incubated with Fc-blocking antibodies. Cells were then washed and incubated for 90 min with H-2D(b):Ig preloaded with a NS3-derived major histocompatibility complex (MHC) I peptide (eg, NS3 cytotoxic T lymphocyte (CTL) epitope with the amino acid sequence APPPSWDAM, H-2D^b). Thereafter, cells were washed and incubated for 30 min with a PE-conjugated rat antimouse IgG1 antibody. Cells were then washed and incubated for 30 min with APC-conjugated rat antimouse CD19 and FITC-conjugated rat antimouse CD8 antibodies. A total of 150 000 events from each sample were acquired on a FACSVerse flow cytometer (BD Biosciences) and analysed using the FlowJo V.9.2 software (Ashland, Oregon, USA). The following antibodies were used: antimouse CD16/32 ‘Fc block’ and antimouse CD19-APC ‘clone 1D3’ (BD Biosciences), and antimouse CD8-FITC ‘clone KT15’ (ProImmune).