L-Cystine dose-dependently increased TNF-alpha from CD14+ monocytes with LPS under the amino acid environment of patients with advanced cirrhosis.
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A, Isolated CD14+ monocytes (purity >90%) were cultured at a density of 2.5×105 cells/well in 96-well plates containing in ACM and ACM plus L-Cys (L-Cys : 150 nmol/mL) with 1,000 U/mL M-CSF.
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Effects of β2-microglobulin (β2m) fibrils on RANKL-dependent osteoclast formation.
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Primary human monocytes were incubated with M-CSF and the indicated concentrations of RANKL in the presence of PBS or 10 µg/ml β2m-fibrils for 14 days and their ability to resorb an osteologic substrate was then assessed.
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Cell preparation
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Human peripheral blood was taken from the antecubital vein of healthy volunteers and collected into tubes containing sodium citrate (1% final concentration) to prevent coagulation.
- Leukocyte populations were then isolated by sedimentation with dextran (500,000 M.wt) followed by discontinuous Percoll™ gradient centrifugation, as reported previously +ve monocytes were cultured for 6 days in tissue culture plates in Iscove's Modified ulbecco's Medium (IMM; ) containing penicillin/streptomycin (PPA , 50 U/ml) and 10% heat-inactivated FCS , in the presence of 50 U/ml GM-CSF to generate Mph1, or 25 ng/ml M-CSF to generate Mph2.
- Following stimulation with LPS (E.coli, serotype O127∶B8) (10 ng/ml for 24 h) Mph1 cells secreted IL-12 but not IL-10, while Mph2 secreted IL-10 but little IL-12, confirming macrophage polarization
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Tks5 as a potential mediator of fusion-competent protrusion formation.
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(E and F) Mouse bone marrow–derived macrophages cultured in the presence of 10 ng/ml M-CSF with or without 10 ng/ml RANKL for the indicated times were subjected either to quantitative RT-PCR analysis of the amount of Sh3pxd2a mRNA (normalized by that of Actb mRNA; E) or to immunoblot analysis with the indicated antibodies .
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Tks5 as a potential mediator of fusion-competent protrusion formation.
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(E and F) Mouse bone marrow–derived macrophages cultured in the presence of 10 ng/ml M-CSF with or without 10 ng/ml RANKL for the indicated times were subjected either to quantitative RT-PCR analysis of the amount of Sh3pxd2a mRNA (normalized by that of Actb mRNA; E) or to immunoblot analysis with the indicated antibodies .
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Impaired osteoclast differentiation, maturation and functionality in osteoporotic mice received CD34+ cells.
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Harvested bone marrow was subjected to differentiation towards osteoclasts using M-CSF and sRANKL (n = 5).
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Human macrophage preparations
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Human macrophages were prepared as previously described 6 cells/ml, and seeded in six-well plates in RPMI 1640 medium supplemented with 10% heat-inactivated FBS .
- Macrophages were differentiated from monocytes in the presence of recombinant human M-CSF (50 ng/ml, , ) for 6 to 11 days.
- The purity of the cell preparations was evaluated by flow cytometry and over 85% of CD11b+/CD206+ macrophages were consistently obtained.
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Effect of inositol hexakisphosphate (IP6) on human peripheral blood mononuclear cells (PBMNC) osteoclastogenesis and resorption activity on mature osteoclasts cells derived from human PBMNC.
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RANKL# refers to cells treated with RANKL and also with M-CSF and dexamethasone as described in the Materials & Methods section.
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Effect of inositol hexakisphosphate (IP6) on human peripheral blood mononuclear cells (PBMNC) osteoclastogenesis and resorption activity on mature osteoclasts cells derived from human PBMNC.
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RANKL# refers to cells treated with RANKL and also with M-CSF and dexamethasone as described in the Materials & Methods section.
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Expression of Rho GTPases on protein level.
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Some expression is observed in immature DCs (iDC), while prominent expression is observed in macrophages differentiated with GM-CSF or M-CSF and in osteoclasts derived from monocytes (mono) or DC .
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