NO-dependent effects of leptin on PEPCK-C protein in WAT explants from rats.
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WAT explants were pre-treated or not with L-NAME (1 mmol/L) for 30 min, then exposed or not to either leptin (10 µg/L), SNAP (1 mmol/L) or INF-γ (50 µg/L) for 2 h in KRB medium containing 2% BSA.
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Experiments on intraperitoneal macrophages.
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Mice at 8–12 weeks of age were injected intraperitoneally with 2 mL 3% thioglycollate 3 days before being killed by CO2 asphyxiation.
- Intraperitoneal macrophages were collected by peritoneal lavage with 10 mL sterile, cold PBS.
- Macrophages were purified by adherence to tissue culture plates for 2 h and cultured at 2 × 106 cells/mL.
- Macrophages were either incubated with 2 units/mL recombinant rat interferon-γ (IFN-γ) for 4 h followed by incubation for 24 h with or without 100 ng/mL lipopolysaccharide (LPS) in PBS vehicle, or with 400 μmol/L palmitate prepared as previously described (6/mL) were cultured with 10 ng/mL recombinant interleukin (IL)-4 ( ocky , ) for 48 h. Arginase I activity was monitored via a colorimetric assay (
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Bone marrow (BM) cell cultures
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BM-derived cells were obtained by flushing tibiae and femurs.
- After lysis of red blood cells with lysing solution , the remaining cells were suspended in complete medium (CM; RPMI 1640 supplemented with 10% fetal bovine serum [FBS], antibiotics, sodium pyruvate, L-glutamine, nonessential amino acids, and 2-ME; all from, , ).
- Where indicated, the cells were stimulated with FMS-like tyrosine kinase 3 ligand (Flt3L; 200 ng/ml, , ), anti-4-1BB (5 µg/ml; clone 3H3, rat IgG2a; produced in-house), LPS (10 µg/ml), ODN-2395 (10 µg/ml), PolyI:C (10 µg/ml), TNF-α (5 ng/ml,), IFN-γ (5 ng/ml), anti-CD40 (5 µg/ml, , ), anti-μ (10 µg/ml , , ), or LA-4Ig (10 µg/ml: Bristol-Myers Squibb, , ).
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HBEC support the proliferation of CD4+ and CD8+ T cells.
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For co-culture 1×105 CFSE-labelled donor PBMC were co-cultured or not with a confluent monolayer of either resting or 10 ng/ml TNF+50 ng/ml IFNγ pre-stimulated HBEC cells.
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Materials
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Rat interferon-γ (IFNγ) was from.
- A methylthiazol tetrazolium (MTT) kit was from .
- Culture multi-wells and pipettes were obtained from Orange Scientific .
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Materials
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Rat interferon-γ (IFNγ) was from.
- A methylthiazol tetrazolium (MTT) kit was from .
- Culture multi-wells and pipettes were obtained from Orange Scientific .
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Nova1 KD increases apoptosis under basal condition and following cytokine treatment.
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Primary rat beta cells were exposed to the pro-inflammatory cytokines IL-1β + IFN-γ for 48 h and then collected for mRNA expression analyses.
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