Quantification of secretory ANGPTL4 and IL-8 in the culture medium was achieved by a DuoSet® Human Enzyme-linked immunosorbent assay (ELISA) development kit (DY3458 and DY208) according to the manufacturer's instructions (R&D Systems). Briefly, 100 μL of culture medium was incubated with precoated capture antibody at room temperature in a 96-well microplate. After washing, 100 μL of detection antibody was added for 2 h at room temperature. Then, 100 μL of the working dilution of streptavidin-HRP was added to each well for 20 min under protection from light. After washing, 100 μL of substrate solution was added for 20 min. After adding 50 μL of stop solution, a SpectraMax i3x Microplate Absorbance Reader (Molecular Devices, CA, USA) was used to quantify the optical density at a wavelength of 450 nm.
Spectramax i3x microplate absorbance reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax i3x Microplate Absorbance Reader is a multi-mode microplate reader that measures absorbance in microplates. It is designed to quantify various biochemical and cell-based assays.
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Lab products found in correlation
2 protocols using spectramax i3x microplate absorbance reader
1
Quantification of ANGPTL4 and IL-8 Secretion
2
Quantification of Secretory ANGPTL4 by ELISA
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Quantification of the secretory ANGPTL4 in the culture medium was achieved by a DuoSet human angiopoietin‐like 4 ELISA development kit (DY3458) according to the manufacturer’s instructions (R&D Systems). Briefly, 100 μL culture medium was collected and incubated with precoated capture Ab at room temperature in a 96‐well microplate. After washing, 100 μL detection Ab was added and incubated for another 2 hours at room temperature, and then 100 μL working dilution of streptavidin‐HRP was added to each well and incubated for 20 minutes under protection from light. After washing, 100 μL substrate solution was added and incubated for 20 minutes under protection from light. After adding 50 μL stop solution, the plate was gently tapped to ensure thorough mixing and an SpectraMax i3x Microplate Absorbance Reader (Molecular Devices) was used to immediately quantify the optical density at 450 nm.
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