The sera were heat-inactivated by incubation at 56 °C Trametinib for 30 min to destroy the activity of serum complement. Bacteria were then washed once with PBS, resuspended in 90 μl of gelatin Veronal buffer (Sigma), and incubated in the presence of 10% fresh-frozen normal mouse serum (from BALB/c mice) at 37 °C for 30 min. After another wash with PBS, the samples were incubated with 100 μl of FITC-conjugated goat antiserum to mouse complement C3 (MP Biomedicals) at a dilution of 1:500 on ice for 30 min in the dark. Finally, the bacteria were washed two more times with PBS, resuspended in 1% formaldehyde, and stored at 4 °C in the dark until analysis with a FACSCanto (BD Biosciences). S. pneumoniae strains

( Table 1) were grown in THY to a concentration of 108 CFU/ml (optical density of 0.4–0.5) and harvested by centrifugation at 2000 × g for 3 min. The pellets were washed once with PBS, resuspended in the opsono buffer [26], and aliquots containing 2.5 × 106 CFU were incubated with heat-inactivated anti-PspA 94/01 or 245/00 pooled sera at a final dilution

of 1:8 and 1:16 at 37 °C for 30 min. Sera from mice immunized with Alum were used as control. After another wash with PBS, the samples were incubated with 10% normal mouse sera (NMS) diluted in opsono buffer at 37 °C for 30 min. The samples were then washed once with PBS and incubated with 4 × 105 peritoneal cells (see Section 2.8) diluted in opsono buffer, at 37 °C for 30 min with shaking (220 rpm). The reaction was stopped by incubation on ice for 1 min. Ten fold dilutions of the samples were performed and 10 μl aliquots of each dilution were plated on blood agar plates. The plates learn more were incubated at a 37 °C, 5% CO2 and the pneumococcal CFU recovered counted after 18 h. The slides were prepared by cytospin and stained with Instant-Prov (Newprov, Brazil). Linifanib (ABT-869) Statistical analysis of the final pneumococcal counts in each group was performed by one-way ANOVA

with a Tukey’s Multiple Comparison Test. BALB/c mice were injected i.p. with 10 μg of Concanavalin A from Canavalia ensiformis (ConA, Sigma), sacrificed 48 h after treatment and their peritoneal cavities washed with 5 ml of ice-cold PBS. The peritoneal cells were adjusted to 4 × 106/ml in opsono buffer [27]. The N-terminal regions of 10 family 1 PspAs (5 clade 1 and 5 clade 2) from Brazilian pneumococcal strains (Table 1) were expressed in fusion with a His-tag in competent E. coli strains and purified through Ni2+ affinity chromatography. The SDS-PAGE of the purified recombinant proteins shows that the molecular mass varied from ∼45 to 70 kDa ( Fig. 1). All fragments included portions of the proline-rich region, and PspAs 245/00, P1031, 325/95, P339 and 94/01 also comprised the non-proline block. Polyclonal sera from BALB/c mice immunized with two or three doses of recombinant PspAs were examined by ELISA and showed similar antibody titers (data not show).