Recombinant human m csf

Mononuclear cells from peripheral blood were isolated from the buffy coats. The monocytes were obtained by Ficoll/Percoll-plus gradient (GE Healthcare Bio-Sciences), as previously described [14 (link)]. Human macrophages were obtained by differentiation of monocytes with recombinant human M-CSF-(Peprotech) at 50 ng/mL for 10 days. The purity of the macrophages was tested by CD14 labeling and flow cytometry analysis (average of 94% of CD14 positive cells). Other cell surface markers were also tested (CD64: 75%; CD11b: 95%; see Supplementary Figure  1 available online at http://dx.doi.org/10.1155/2015/942517). All the reagents used for the cell culture were endotoxin-free, as assayed with the Limulus amebocyte lysate test (Cambrex). The workflow to establish the different in vitro models was as indicated in each figure legend.

Peripheral blood mononuclear cells were isolated as described above and added to triplicate wells of 24-well plates at a concentration of 2 X 10⁶ cells/ml for 2h at 37°C to allow monocyte adherence. Non-adherent cells were then washed off gently using PBS, and the wells re-fed with complete medium containing 10 ng/ml recombinant human M-CSF (PeproTech, Rocky Hill, NJ). After 7 days in culture, macrophages were fully differentiated from monocytes and then used in assays to examine the impact of LTC treatment on activation and bacterial killing.

THP-1 cells (purchased from National Infrastructure of Cell Line Resource, http://www.cellresource.cn/) were maintained in RPMI1640 media supplemented with L-glutamine, 1% penicillin and streptomycin, β-mercaptoethanol (Gibco, 2169148, 0.055 mM) and 10% fetal bovine serum (FBS, Gibco) at 37°C under a humidified, 5% CO₂ atmosphere (16 (link)). THP-1 cells were differentiated to M0 macrophages by treatment with 25 nM phorbol 12-myristate 13-acetate (MCE, HY-18739) for 48 h, washed and incubated with normal RPMI1640 media for 24 h, and then incubated with recombinant human GM-CSF (50 ng/ml, Peprotech, 300-03) for 96 h. For M2 polarization, 50% of complete RPMI1640 medium was added, and it was incubated for 48 h. Then the M2 macrophage was obtained by removing the culture medium and culturing cells for an additional 48 h in M2 medium with recombinant human M-CSF (100 ng/ml, Peprotech, 300-25) (17 (link)).

NOD/SCID mice were subcutaneously injected with 0.5 mg/kg recombinant human M-CSF (PeproTech, 300-25) before adoptive transfer [20 (link), 21 (link)]. For monocyte recruitment, human MSCs pretreated with MRF siRNA or the negative control were intraperitoneally injected into the mice. Twelve hours later, 1 × 10⁷ human CD14+ monocytes labeled with CFSE were intravenously injected into each mouse. After 24 h, the adoptively transferred mice were sacrificed, and peritoneal lavage fluid and spleen cells were collected for flow cytometry analysis. For macrophage polarization, human MSCs were pretreated with MRF siRNA or negative control, human macrophages were activated by human M-CSF and then stimulated with LPS and recombinant human IFN-γ to induce M1 polarization or with recombinant human IL-4 and IL-10 to induce M2 polarization, similar to the in vitro induction of macrophage polarization. Then, 1 × 10⁵ human MSCs and 1 × 10⁶ macrophages labeled with CFSE were simultaneously injected into the abdominal cavity of NOD/SCID mice. Two days later, transplanted mice were sacrificed, and peritoneal lavage fluid was collected to detect the MFI of human HLA-DR and human CD206 in CFSE-labeled cells via flow cytometry.

CD14⁺ cells were purified from PBMCs using the EasySep™ Human Monocyte Enrichment Kit (StemCell Technologies) and differentiated into macrophages with 100 ng/mL recombinant human M-CSF (Peprotech). After 6 days cells were harvested, seeded in 96 well plates and rested overnight. Cells were primed with 20 ng/mL human recombinant IFNγ (R&D systems) for 24 h, followed by 3 μM MEDI9197 or 20 ng/mL LPS (Invivogen) for 24 h. Supernatants were analysed for IL-12p70 using the human IL-12p70 ELISA Duoset kit (R&D).