Thermo multiskan spectrum plate reader

Serum samples were assayed for IL-6 concentration using commercially available human IL-6 enzyme linked immunosorbent assay kits (Quantikine, R&D systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Creatine kinase (CK) activity was assayed using a commercially available CK assay (Catachem Inc., Connecticut, NE, USA). Ten μL blood serum were loaded onto a 96-well plate. The CK reaction reagent and diluent (Catachem) were prepared as per the manufacturer’s instructions and added to the samples and the change in absorbance monitored continuously over 20 min in a Thermo Multiskan Spectrum plate reader (Thermo Fisher Scientific. Waltham, MA. USA) at a wavelength of 450 nm (IL-6) and of 340 nm (CK activity).

For data confirmation of the array and quantification of the angiogenesis-related proteins with an average pixel intensity of ≥1.0³ arbitrary intensity units^{58 (link)} as defined with the angiogenesis array, enzyme-linked immunosorbent assays (ELISA) were performed. Levels of Vascular Endothelial Growth Factor A (VEGF A), urokinase-type Plasminogen Activator (uPA), Insuline-like Growth Factor Binding Protein (IGFBP-3), Interleukin 8 (IL-8), Monocyte Chemoattractant Protein 1 (MCP-1), Pentraxin-3 (PTX-3, also known as TSG-14), Plasminogen Activator Inhibitor 1 (PAI-1 also known as Serpin E1), Pigment Epithelium-derived Factor (PEDF, also known as Serpin F1), Thrombospondin-1 (TSP-1) and TIMP Metallopeptidase Inhibitor (TIMP-1) were measured in the CM of ES and Control groups for each respective time point, using Duoset ELISA kits (R&D Systems Inc.) according to the manufacturer’s instructions. The OD’s were measured at a wavelength of 450 nm using a Thermo Multiskan Spectrum plate reader and SkanIt software (Thermo Fisher Scientific).

The EIA was performed as previously described by Finkenwirth et al. [35 (link)]. Briefly, a 96 well microtiter plate was coated with affinity purified goat anti-rabbit IgG antibody and duplicates of 20 μl standards (0.4–400 μg/well) and fecal extracts, prepared in assay buffer (50 mM Na₂HPO₄/Na₂HPO₄, 0.15 M NaCl, 0.1% BSA, pH 7.4), were added simultaneously into the respective wells along with 100 μl of PGFM-HRP (horse radish peroxidase) conjugate which was diluted in assay buffer (1:20,000). Then, 100 μl of PGFM-specific antiserum (diluted 1:20,000 in assay buffer) was added to all the wells, except the blank, and the plates were incubated overnight at 4°C. The plates were washed the following day with washing solution. Thereafter, 150 μl of substrate solution (1.2 mM H₂O₂, 0.4 mM 3,3’,5,5’-tetramethylbenzidine in 10 mM sodium acetate, pH 5.5) was added to each well and incubated in the dark. The reaction was stopped with 50 μl of 4N H₂SO₄ and the color[US English] intensity was measured at 450 nm in an ELISA reader (Thermo Multiskan Spectrum Plate Reader, version 2.4.2, Thermo Scientific, Finland.