Rat sera

The infected BMDCs were washed three times with ice-cold PBS and blocked on ice for 30 min with super block solution containing 10% mouse sera (Sigma Aldrich), 10% rat sera (Sigma Aldrich), 10% goat sera (Gibco Invitrogen), and 2.4G2 monoclonal antibody (10 μg/ml, Invitrogen). The blocked BMDCs were stained with APC-Cy7-conjugated anti-CD11c (clone N418; BD Pharmingen), FITC-conjugated anti-I-A^b (clone AF6-120.1; BD Pharmingen), PE conjugated anti-CD40 (clone 3/23; BD Pharmingen), APC-conjugated anti-CD80 (clone 16-10A1; eBioscience) and PE/Cy7-conjugated anti-CD86 (clone GL-1; BioLegend) for 30 min on ice and washed three times with cold PBS. The stained BMDCs were resuspended in FACS buffer (PBS with 1% bovine serum albumin and 1 mM EDTA). The BMDCs were analyzed by flow cytometry (BD LSRFortessa). The data were analyzed with FlowJo software version 10.1 (FlowJo, Ashland, OR, USA).

Lymphocytes collected from the spleens of immunized mice were cultured for 18–20 h in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS, 50 nM β-mercaptoenthanol, 100 units/ml penicillin/streptomycin, 2 mM l-glutamine (Welgene), and 10 μg/ml of purified ScaA in 24-well, flat-bottomed culture plates (5 × 10⁶ cells/well). After incubation, GolgiPlug (BD Biosciences) was added for 6 h. Lymphocytes were washed three times with ice-cold FACS buffer (PBS containing 1% bovine serum albumin and 1 mM EDTA). Cells were blocked on ice for 30 min with ultra-block solution containing 10% rat sera, 10% hamster sera, 10% mouse sera (Sigma) and 10 μg/ml of 2.4G2 monoclonal antibody (BD Pharmingen). Cells then stained with FITC-conjugated CD4 or PE.cy7-conjugated CD8 antibodies (BD Pharmingen) for 30 min at 4 °C. After surface CD4 or CD8 staining, cells were washed three times with ice-cold FACS buffer and subjected to intracellular cytokine staining using the Cytofix/Cytoperm kit according to the manufacturer's instructions (BD Biosciences). Intracellular interferon-γ (IFN-γ) was stained using an APC-conjugated anti-IFN-γ antibody (BD Pharmingen) for 30 min at 4 °C. The stained cells were analyzed with a FACS LSRII flow cytometer (BD Biosciences). Data were analyzed by FlowJo software version 8.8.6 (FlowJo).