Cpg oligodeoxynucleotides odn

Freshly isolated islets from Tlr9^+/+ NOD mice (5-week-old females) were cultured overnight with the TLR9 antagonist CpG- oligodeoxynucleotides (ODN) (2088; Invivogen, San Diego, CA, USA) or control CpG-ODN (Invivogen), both at 10 μg/ml. After extensive washing, a single-cell suspension was prepared as described earlier and stained with fluorochrome-conjugated monoclonal antibodies to CD45, CD140a and FluoZin-3-AM before analysis by flow cytometry. Another set of freshly isolated islets from Tlr9^+/+ NOD mice was used for insulin release assay, after overnight culture in the presence of the TLR9 antagonist CpG-ODN or control CpG-ODN.

Isolation and culture of primary bone marrow cells were performed, as previously described [36 (link),37 (link)]. Briefly, bone marrow cells were isolated from the long bones of 16-week-old female mice and cultured on Petri dishes for 2 h at 37 °C under 5% CO₂. Non-adherent bone marrow cells were re-seeded on culture plates at a density of 1 × 10⁶ cells/mL and then incubated for 2 days in α-minimum essential medium (α-MEM) containing 10% heat-inactivated FBS and 25 ng/mL recombinant mouse macrophage colony-stimulating factor (M-CSF; PeproTech). After the 2-day pre-culture, the yielded bone marrow-derived macrophages were stimulated with Toll-like receptors ligands, such as lipopolysaccharide (LPS, Sigma-Aldrich), polyinosinic-polycytidylic acid sodium salt (Poly(I:C)), single-strand RNA (ssRNA), and CpG oligodeoxynucleotides (ODN, all from InvivoGen, San Diego, CA, USA), for 6 h in the presence of M-CSF. RNA samples were isolated from bone marrow-derived macrophages at the indicated time points and subjected to gene expression analysis.

As for differentiation of Raw264.7 mouse macrophages, Raw264.7 were stimulated for IL-4 (20 ng/ml, Novoprotein, China) and IL-13 (20 ng/ml, Novoprotein, China) or LPS (250 ng/ml, Sigma) and IFN-γ (20 ng/ml, Sigma) for 24 h. TLR9 antagonist CpG-oligodeoxynucleotides (ODN) (2088; Invivogen, San Diego, CA, United States) or control CpG-ODN (Invivogen) was co-cultured with stimulants to determine the effects of TLR-9 on macrophages.