Bone marrow derived murine monocytes were isolated from 6-week-old C57BL/6 mice (Charles River Laboratories) as described previously [21 (link)]. Briefly, tibias and femura were extracted from the mice, and the bone marrow flushed using IMDM (Invitrogen). The bone marrow isolates were suspended in IMDM with PSF, layered in Lympholyte M (Accurate Chemicals) and centrifuged per the manufacturer instructions. The portion containing mononuclear cells was collected and resuspended at 106 cells/ml in expansion medium, containing IMDM, 20% fetal bovine serum (FBS), 2mM L-glutamine, PSF, 1.5 ng/ml human macrophage colony stimulating factory (R&D systems) and 100 ng/ml huFLT-3 (R&D systems). The cells were plated in non-tissue culture treated polystyrene flasks at 1.7×105 cells/cm2 and cultured for 10 days. Cells were then seeded onto acellular or MSC-laden hydrogels at 2.5×105 macrophages/cm2 in MSC culture medium. Hydrogel samples were cultured in the absence or presence of 1 µg/ml lipopolysacharid (LPS, E. coli, Sigma-Aldrich) for 4, 8 or 24 hours.
Iscove modified dulbecco media (imdm)
Manufactured by R&D Systems
IMDM (Iscove's Modified Dulbecco's Medium) is a cell culture medium formulated for the growth and maintenance of a variety of cell types. It provides a balanced salt solution, amino acids, vitamins, and other nutrients required for cell proliferation. The core function of IMDM is to support the in vitro cultivation of cells, including those derived from human, mouse, and other sources.
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Lab products found in correlation
Iscove modified dulbecco media (imdm), by Thermo Fisher Scientific (2 mentions)
E coli lps, by Merck Group (1 mentions)
C57bl 6 mice, by Charles River Laboratories (1 mentions)
Huflt 3, by R&D Systems (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Hifcs, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
Inf γ, by R&D Systems (1 mentions)
Gm csf, by R&D Systems (1 mentions)
2 protocols using iscove modified dulbecco media (imdm)
1
Isolation and Differentiation of Murine Monocytes
2
Macrophage Differentiation and Activation
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Monocytes were cultured in IMDM (Gibco) with 2% autologous human AB serum (AS; Sigma Aldrich) overnight before use. MDM were generated by culturing the monocytes for 5 days in IMDM (2% AS) with 5 ng/ml GM-CSF and 20 ng/ml IL-6 (R&D Systems) to drive differentiation into a macrophage phenotype36 (link). During this period, no media was removed, but an additional volume of fresh media (25% of the total volume) containing 2% AS but no cytokines was added on day 3. To induce inflammatory MDM, INFγ (20 ng/ml; R&D Systems) was added 24 h before assay. Immediately before assay the cells are washed with HBSS (Gibco). AM were cultured in IMDM with 2% AU for a minimum of 2 days before treatment. Live neutrophils (from both blood and BAL) were re-suspended in IMDM without serum and used immediately in the appropriate co-culture assay. BL2 Burkitt’s lymphoma cells and Jurkat cells were cultured in RPMI (Gibco) with 10% heat inactivated foetal calf serum (hiFCS; Gibco) and maintained at a density of 0.3–1 million per ml. All cells were cultured using standard culture conditions: 37 degrees with 5% CO2.
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