Lipopolysaccharides lps from escherichia coli o111 b4

Ibrutinib (Cambridge BioScience, UK); LPS (Lipopolysaccharides from Escherichia coli O111:B4, Sigma, UK), general anaesthetic used.

Bovine macrophages (4 × 10⁵ cells/mL) were inoculated with live U. diversum strains ATCC49782 (10⁶, 10⁵, and 10⁴Ureaplasma/mL) and CI-GOTA (10⁵, 10⁴, and 10³Ureaplasma/mL), as well with inactivated U. diversum strains ATCC49782 (inactivated from 10⁶Ureaplasma/ml) and CI-GOTA (inactivated from 10⁵Ureaplasma/mL). Infected monolayers were incubated for 6, 12, and 24 h at 37°C in 5% CO₂. Regarding the extracted-lipoproteins, macrophages were inoculated with UdLAMPs from both strains at concentrations of 2.0, 1.5, 1.0, and 0.5 μg/mL for 2, 6, and 12 h. Macrophages inoculated with PBS and 100 ng/mL of lipopolysaccharides (LPS - Lipopolysaccharides from Escherichia coli O111:B4, Sigma-Aldrich, Brazil) served as negative and positive controls, respectively. At each time, cells were collected, suspended in RNAlater^TM (Invitrogen, Brazil) and frozen at −70°C for subsequent mRNA extraction. All experiments were performed in triplicate with three independent repetitions (n = 9).

CD19 + cells were isolated from mouse spleens using Microbeads anti-CD19 + (Miltenyi, #130–121-301) and the purity of cells was assessed by flow cytometry using an antibody against CD19 (CD19 FITC (BD, clone CL1D3, #557,398). For western blot analysis, 2 × 10⁶ cells were seeded in 6-well plates and stimulated for 1 h. For flow cytometry analysis and RT-qPCR 1 × 10⁶ cells were seeded in 12-well plates and stimulated for 3 days. The following TLR agonists were used: the TLR4 agonist LPS (Lipopolysaccharides from Escherichia coli O111:B4, Sigma, #L4130) (1 µg/ml), the TLR9 agonist CpG 1826 (TriLink Biotechnologies, #I66-G01A) (5 µg/ml), the TLR3 agonist Poly I:C (Polycytidylic-inosinic acid potassium salt, Sigma, #P1038) (5 µg/ml). After stimulation cells were collected and washed in PBS. Dry pellets were stored at -80 °C for western blot analysis, while fresh samples were stained with the different antibodies for flow cytometry analysis of activation (FITC-CD19, BD, clone CL1D3, #557,398 and PE-CD86, eBioscience, clone GL1, #12–0862-82).
To evaluate TLR9-MYD88 cascade in OPL239/OPL241 DLBCL cell lines, 1,5 × 10⁶ cells were seeded in 6-well plates and stimulated with 5 μg/ml CpG for 30 min for western blot and 3 h for RT-PCR. Cells were washed in PBS and dry pellet stored at -80 °C for analysis.