Flow cytometry

The mice were inoculated with 4T1 cells and were treated with ME49Δgra5 parasites or PBS as described above. At 21 dpi, six mice from each group were euthanized to harvest the spleens which were homogenized and filtered to get the spleen single-cell suspension, and red blood cells were lysed with erythrocyte lysis buffer. The concentration of splenocytes was adjusted to 1 × 10⁶ cells/ml with PBS after washing with RPMI-1640 medium twice. Then, the single-cell suspensions were incubated with the antibodies (Univ, China) of panel 1—percp-cy5.5 anti-mouse CD45, BV510 anti-mouse CD3e, APC anti-mouse CD4, FITC anti-mouse CD8a, PE-cy7 anti-mouse CD19, and PE anti-mouse CD49b—and panel 2—percp-cy5.5 anti-mouse CD45, PE anti-mouse F4/80, FITC anti-mouse CD11b, and APC anti-mouse CD11c—at 4°C for 30 min following the manufacturer’s guidance. The analyses of splenocytes were performed with fluorescence profiles using a FACScan flow cytometer (BD Biosciences, USA).