Human il 1β il 1f2 duoset elisa development kit

Cell culture media was collected and soluble factors expression was analysed 24 h after initiation of cell culture. The levels of IL6, IL8/CXCL8, CCL2, IL1B, IL2, IL4, IL12, TNFα and IFNγ secreted into the growth medium were measured using Human IL-6 DuoSet ELISA Development Kit (R&D System, Wiesbaden, Germany), Human IL-8 Standard ABTS ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human CCL2/MCP-1 DuoSet (R&D System), Human IL-1β/IL-1F2 DuoSet ELISA Development Kit (R&D System, Wiesbaden, Germany), Human IL-2 ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human IL-4 Standard ABTS (PeproTech, Rock Hill, NJ, USA), Human IL-12 ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human TNFα ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), Human IFN-γ ELISA Development Kit (Peprotech, Rock Hill, NJ, USA), and Human Standard ABTS ELISA Development Kit (Peprotech), respectively. The ELISA analysis was performed using high binding ELISA plates (Greiner BioOne) at RT according to the manufacturer’s instructions. Optical density was measured using photospectrometer Spectramax 340 PC (Molecular Devices) at the wavelength 450 nm.
The represented data show values of two independent analyses normalized to the levels of cytokine expression for cells grown without GAIN scaffolds.

Expression of IL-1β in tumour conditioned media and macrophage conditioned media was conducted using a human IL-1β/IL-1F2 duoset ELISA development kit (R&D Systems) according to the manufacturers’ protocols. Briefly, a 96-well plate was coated with capture antibody overnight and the plate was washed and blocked with bovine serum albumin. Samples and IL-1β recombinant protein standard were added to the plate for 2 h. This was followed by detection antibody for 2 h and streptavidin-HRP for 20 min, with each step preceded by a wash. Colour change was achieved by the addition of substrate followed by a stop solution, and the plate was read at 450 nm on a BMG Fluostar Optima (BMG Labtech).