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MB Sample ID: SA177934

Local Sample ID:	UT5
Subject ID:	SU002000
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Treatment:

Treatment ID:	TR002012
Treatment Summary:	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), anti-C7 antibody with NHS (previously incubated for 30 min, on ice) as a negative control for MAC formation, 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.

Treatment ID:

TR002012

Treatment Summary:

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), anti-C7 antibody with NHS (previously incubated for 30 min, on ice) as a negative control for MAC formation, 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.