Multiplex immunoassays (LXSAHM-03, R&D Systems, Mineapolis, MN, USA) were used to investigate fibroblast expression levels of interleukin-6 (IL-6), interleukin-1 receptor antagonist (IL-1RA), and platelet-derived growth factor alpha (PDGF-α) in cell lysates following the manufacturer’s instructions. The Luminex MAGPIX system with xPONENT 4.3 software (Luminex Corporation, Austin, TX, USA. A DiaSorin Company) instrument was used for analyses of the multiplex immunoassays (using Belysa ® Immunoassay Curve Fitting Software (Merck KGaA, Darmstadt, Germany)). The ELISA assay (Human TGF beta 1 Elisa kit, ab108912, Abcam, Cambridge, UK) was further used for determining levels of transforming growth factor beta 1 (TGF-β1). Absorbance at 450 nm was measured using a 96 Plate Reader (Enzo Life Sciences, Inc., East Farmingdale, NY, USA) and additional background subtraction was done at 570 nm (analysis with Byonoy Software, Byonoy GmbH). Total protein quantification was done with a bicinchoninic acid assay (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) and these data were used for normalization of the data.
96 plate reader
Manufactured by Enzo Life Sciences
Sourced in United States
The 96 Plate Reader is a versatile laboratory instrument designed for high-throughput optical analysis. It is capable of performing absorbance, fluorescence, and luminescence measurements in standard 96-well microplates. The device features a robust and user-friendly interface, providing researchers with a reliable tool for a wide range of applications in life science research.
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2 protocols using 96 plate reader
1
Multiplex Immunoassay for Fibroblast Secretome
2
Fibroblast Cytokine and Growth Factor Expression
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Fibroblast expression of pro-inflammatory cytokines and growth factors and were quantified in lysates (see “Extracellular matrix protein expression” for a description of how lysates were collected) with Multiplex immunoassay (Interleukin-6 (IL-6), Interleukin-1 receptor agonist (IL-1RA), Luminex Discovery Assay, LXSAHM-03, R&D systems) and ELISA assays (transforming growth factor-beta (TGF-β1), Human TGF beta 1 ELISA kit, ab108912, Abcam) following manufactures instructions. Analyses was performed on Luminex (MAGPIX system with xPONENT 4.3 software (Luminex Corporation. A DiaSorin Company)) instrument for the Multiplex immune assay (analysis performed with Belysa® Immunoassay Curve Fitting Software (Merck, KGaA) and on the absorbance 96 Plate Reader (Enzo Life Sciences, Inc) measured at 450 nm and background subtraction at 570 nm (analysis performed with Byonoy software (Byonoy GmbH). Total protein quantification was done with bicinchoninic acid assay (Sigma-Aldrich), and these data were used for normalization of the data.
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