Commercially available Enzyme Linked Immunosorbent Assay (ELISA) kits for measuring apelin levels in human and mouse serum, cell culture medium, cell lysate and cholangiocyte supernatant were purchased from Sigma-Aldrich (16 (link)). The commercially available kits for measuring ROS levels in liver samples and cultured cells were purchased from Abcam (Cambridge, MA) and Cell Biolabs, Inc. (San Diego, CA). ELISA and ROS kits were performed according to the manufacturer’s instructions.
Enzyme linked immunosorbent assay elisa kit
Manufactured by Merck Group
Sourced in United Kingdom
The Enzyme Linked Immunosorbent Assay (ELISA) kits are laboratory equipment used to detect and quantify specific substances in a sample, such as proteins, hormones, or antibodies. The kits utilize enzyme-labeled antibodies to bind to the target analyte, allowing for the measurement of its concentration through a colorimetric or fluorometric reaction.
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5 protocols using enzyme linked immunosorbent assay elisa kit
1
Measuring Apelin and ROS Levels
2
Quantifying VEGF Secretion in BMSC-Chondrocyte Co-culture
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To evaluate secretory VEGF from all groups of cells, we set the following groups: (a) BMSC: 1 × 105 hBMSCs transfected with empty lentivirus; (b) C + BMSC: A total of 1 × 105 hBMSCs transfected with empty lentivirus and 3 × 104 chondrocytes were cultured; (c) CB + BMSC: A total of 1 × 105 hBMSCs transfected with empty lentivirus and chondrocyte bricks formed from 3 × 104 chondrocytes were coseeded; (d) CB + BMSCVEGF−: A total of 1 × 105 VEGF‐silencing hBMSCs and chondrocyte bricks formed from 3 × 104 chondrocytes were coseeded. These cells were seeded in 6‐well plates and cultured for 24 hours; the medium was replaced with 2 ml DMEM containing 10% FBS for an additional 48 hours. Collected medium was centrifuged (2500 rpm for 3 minutes) to remove cell debris and then used for experiments. Enzyme‐linked immunosorbent assay (ELISA) kits (Sigma‐Aldrich) were used to quantify VEGF concentration in medium from each group according to the manufacturer's instructions. Additionally, seeded cells from each group were harvested for protein extraction and analysis by Western blot.
3
Quantifying Secreted and Cellular SCF
4
Glucose-Stimulated C-Peptide Secretion Assay
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The IPCs were assessed the function by glucose-stimulated C-peptide secretion assay (GSCS)24 (link),38 (link),69 (link),70 (link),138 (link). The IPCs were incubated in normal KRH bicarbonate (KRBH) buffer (pH 7.4) for 1 h as basal control, then in 5.5 mM of glucose anhydrous (Sigma) in KRBH for the next 1 h and finally in 22 mM glucose anhydrous in KRBH for 1 h. Enzyme-linked immunosorbent assay (ELISA) kit (Millipore) was used for detecting the generated C-peptide level according to the manufacturer’s protocol. Total DNA (ng) and stimulation time (mins) were used to normalization.
5
Insulin Secretion Assay in MIN-6 Cells
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To evaluate the function of MIN-6 cells, the amount of insulin secreted from non-coated and coated cells in response to changes in glucose concentration was measured. Briefly, samples were washed and pre-incubated in Krebs-Ringer bicarbonate buffer (KRBH: 125 mM NaCl, 1.2 mM MgSO4, 1.2 mM CaCl2, 22 mM NaHCO3, 10 mM HEPES, 1.19 mM KH2PO4) + 0.1% BSA with glucose concentration of 2 mM for 2 h. Then, samples underwent a static incubation for 1 h with low (2 mM) glucose concentration in KRBH + 0.1% BSA followed incubation for a further hour in high (20 mM) glucose concentration KRBH + 0.1% BSA as the stimulator. For each incubation period the cultured supernatant was collected for insulin assay and diluted to the appropriate range based on the assay standard curve. The secreted insulin was measured using an Enzyme-linked immunosorbent assay (ELISA) kit (Millipore, UK), and the colorimetric reaction was quantified using a plate spectrophotometer (GloMax-Multi + Mictoplate Multimode reader, Promega, USA) at a wavelength of 450 nm.
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