Complete rpmi medium

Blood samples were taken from the temporal plexus of mice 0, 12, and 28 days after priming and 3 and 10 days after boosting. Samples were incubated for 30 min at 37°C and centrifuged at 1,200 × g at 4°C for 10 min for collecting sera that were stored at −80°C. Draining lymph nodes (sub iliac, medial, and external) and spleens were collected 7 and 28 days after priming, and 3 or 10 days after boosting. Samples were mashed onto 70 µm nylon screens (Sefar Italia, Italy) and washed two times in complete RPMI medium [RPMI (Lonza, Belgium), 100 U/ml penicillin/streptomycin, and 10% fetal bovine serum (Gibco, USA)]. Samples were treated with red blood cells lysis buffer (eBioscience, USA) and counted with cell counter (Bio-Rad).

Thioglycolate elicited mouse peritoneal macrophages were isolated in ice-cold Dulbecco’s PBS and allowed to adhere onto sterile coverslips on a 24-well cell culture plate containing complete RPMI medium (Lonza, Basel, Switzerland). One hour later, the cells were washed with PBS to remove non-adherent cells. Similarly, RAW 264.7 cells were also seeded in a cell culture plate and 10 µg/ml of the recombinant PbTIP was incubated with peritoneal macrophages and RAW 264.7 cells for 1 h. After incubation, the cells were washed with an excess of PBS and fixed with 4% paraformaldehyde (PFA), followed by IFAs to decipher the surface binding of PbTIP.