Cluster tubes

Bead-based LEGENDplex immunoassay (BioLegend, London, UK) was used for the quantification of plasma cytokines and cytokines in cell culture supernatants. Procedures were performed according to the manufacturer’s protocol using cluster tubes (ThermoScientific, Loughborough UK). 12.5 μl of plasma from EDTA blood samples was used diluted 1:2 in assay buffer. Supernatants from bmMΦ were used non-diluted. Analyses were performed using a BD Accuri C6 (BD Biosciences, San José, USA) and LEGENDplex analysis software (BioLegend, San Diego, USA).

100μl of fresh or frozen whole blood from rhesus macaques were placed into cluster tubes (Thermo Fisher Scientific) and incubated with either the anti-Mamu-A1*001 Fab-containing bacterial lysate, MHC class I-specific antibody W6/32 (positive control), or non-specific mouse IgG1 (negative control) for 30 minutes at room temperature. The red blood cells were lysed by incubating with BD FACS Lyse solution (BD Biosciences) for 10 minutes at room temperature. The cells were washed twice with FACS buffer. Cell-bound anti-Mamu-A1*001 Fab fragments were detected with anti-HA FITC and control antibodies with goat anti-mouse Ig FITC secondary antibodies. After a 30-minute incubation at room temperature the cells were washed twice with FACS buffer, fixed with 2% paraformaldehyde (PFA) and stored at 4°C until being acquired on a LSR II flow cytometer. The data was analyzed using FlowJo software.

Plasma cytokines were quantified by bead-based LEGENDplex immunoassay (BioLegend, London, UK) according to the manufacturer’s protocol using cluster tubes (ThermoScientific, Loughborough UK). 12.5 μl of plasma from EDTA blood samples was used diluted 1:2 in PBS. Supernatants from bmMΦ were used non-diluted. Analyses were performed using a BD Accuri C6 (BD Biosciences, San José, USA) and LEGENDplex analysis software (BioLegend, San Diego, USA).