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MB Sample ID: SA177926
| Local Sample ID: | MAC3 |
| Subject ID: | SU002000 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
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Collection:
| Collection ID: | CO001993 |
| Collection Summary: | PBMCs were isolated from healthy human blood cones (supplied by GlaxoSmithKlein) by gradient centrifugation. The PBMC layer was collected and monocytes isolated using CD14+ beads (Miltenyi Biotech) according to supplier’s protocol. For proteomics and metabolomics analysis, frozen human primary monocytes were used (supplied by Lonza). Purified monocytes were plated at relevant cell concentration for experiment and treated with growth factor GM-CSF (5 ng/ml) and cultured in RMPI-1640 (Life Technologies) with 5% FCS and 2mM L-glutamine for 6 days, at 37 °C, 5% CO2 to allow differentiation. All human biological samples were sourced ethically, and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol. On day 6, cells were washed once with treatment media RPMI-1640 with 2mM L-Glutamine and sensitised to complement attack by adding 7 µg/ml of anti-CD55, anti-CD59 and anti-HLA antibodies for 50 min at 37 °C, 5% CO2. Antibody-sensitised cells were exposed to normal human serum (NHS), or non-sensitised cells with NHS alone at 37 °C, 5% CO2 for the indicated amount of time. Subtlytic doses of MAC were characterised as <20% cell death (Campbell, Daw et al. 1979, Reid, Cooke et al. 2012). Alternatively, MAC attack was induced using human purified proteins C5b6-9 for the extracellular H2O2 assay. Cells were incubated with anti-CD59 for 50 min at 37 °C, 5% CO2. Antibody-sensitised cells were exposed to purified protein C5b6 for 10 min at room temperature, followed by addition of purified C7 for 15 min at 37 °C, 5% CO2. C8 and C9 were then added sequentially and left at 37 °C, 5% CO2 for the indicated amount of time. C7, C8 and C9 were added in a molar excess to the C5b6 concentration. |
| Sample Type: | human monocyte-derived macrophages (hMDM) |
