Primary microglia from mice—chosen for species-specificity of available antibodies—were plated at densities of 10,000 cells/100 μl/well (96 well-plate format) in culture medium. Cells were treated for 24 h, and cell media was extracted and subjected to a calreticulin ELISA (Abbexa, Cambridge, UK) or galectin-3 ELISA (R&D Systems, Minneapolis, USA) as per manufacturer's instructions. Absorbances at 450 nm were measured using a FLUOstar Optima plate reader (BMG Labtech, Ortenberg, Germany) and represented as protein concentration calculated against a standard curve.
Galectin 3 elisa
Manufactured by R&D Systems
Sourced in United Kingdom
The Galectin-3 ELISA is a quantitative sandwich enzyme-linked immunosorbent assay designed for the measurement of Galectin-3 levels in human serum, plasma, and cell culture supernatants. The assay utilizes a monoclonal antibody specific for Galectin-3 that has been pre-coated onto a microplate. Samples and standards are pipetted into the wells, and Galectin-3 present in the sample is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for Galectin-3 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Galectin-3 bound. The intensity of the color is measured.
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2 protocols using galectin 3 elisa
1
Quantifying Secreted Proteins from Primary Microglia
2
Lymphatic Exudate and Plasma Protein Analysis
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Total protein content of lymphatic exudate and plasma was assessed by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Most of the cytokines were assessed by ELISA analysis according to the manufacturer’s instructions. ELISAs used were the following: galectin-3 ELISA (DGAL30; R&D Systems), M-CSF (CSF-1) ELISA (DMC00B; R&D Systems), S100B ELISA (KA0037; Abnova), S100A8 (DY4570; R&D Systems), MMP-2 ELISA (DY902; R&D Systems), MMP-9 ELISA (BMS2016/2; Affimetrix), VEGF-C (DuoSet DY293B; R&D Systems), and VEGF-D (DuoSet DY622; R&D Systems). Lactate dehydrogenase activity was measured using the LDH Activity Assay Kit (MAK066; Sigma-Aldrich). IL-6, IL-8, TNF-α, IL-1β, IL-4, and IL-10 were analyzed using the Proinflammatory Panel 1 Kits of Mesoscale (MSD Multi-Spot Assay System).
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