Transient Plasmid Transfection Assay

Transient plasmid transfection was carried out as previously described ^{42 (link)} using constitutively active TRIF, MAVS or the pUNO1 control vector (Invivogen, France), 0.25μg CXCL8 or IFN-β reporter were kindly provided by Dr MR Edwards (Imperial College, London), 0.1μg of Renilla plasmid (Promega, Southampton, UK) and Superfect transfection reagent (Quiagen, Manchester, UK). At 5h post transfection, cells were washed and treated with SAPS at the indicated concentrations. Lysates were harvested at 24h, and luciferase measured according to the dual luciferase protocol (Promega, Southampton, UK) using a Berthold luminometer (Berthold Technologies, Herts, UK) with data expressed as ratios of firefly over renilla luciferase.

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Stokes C.A., Kaur R., Edwards M.R., Mondhe M., Robinson D., Prestwich E.C., Hume R.D., Marshall C.A., Perrie Y., O'Donnell V.B., Harwood J.L., Sabroe I, & Parker L.C. (2016). Human rhinovirus-induced inflammatory responses are inhibited by phosphatidylserine containing liposomes. Mucosal Immunology, 9(5), 1303-1316.

Publication 2016

pUNO1 control vector Renilla plasmid Superfect transfection reagent dual luciferase protocol Berthold luminometer

Cells Cxcl8 Firefly Luciferase Mavs Plasmid Promega Renilla Renilla luciferase Saps Transfection Transient

Corresponding Organization : University of Sheffield

Other organizations : Aston University, Imperial College London, Cardiff University, University of Cambridge

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Variable analysis

independent variables

TRIF (constitutively active)
MAVS (constitutively active)
PUNO1 control vector
CXCL8 reporter
IFN-β reporter
SAPS (at indicated concentrations)

dependent variables

Luciferase activity (firefly/renilla ratio)

control variables

Transfection reagent (Superfect)
Renilla plasmid (0.1μg)
Incubation time (5h post-transfection, 24h lysate harvest)

positive controls

CXCL8 reporter
IFN-β reporter

negative controls

PUNO1 control vector

Annotations

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