The quantity of IL-1β in the supernatant of BV2 cells stably expressing NC and shMEF2D was tested using mouse enzyme-linked immunosorbent assay kits (R&D Systems, Minnesota, USA) according to the manufacturer’s procedures.
Mouse enzyme linked immunosorbent assay kit
Manufactured by R&D Systems
Sourced in United States
Mouse enzyme-linked immunosorbent assay (ELISA) kits are laboratory tools used to detect and measure specific proteins or analytes in mouse biological samples. These kits employ antibody-based techniques to quantify the target analyte. The core function is to provide a standardized and reliable method for the quantitative analysis of mouse proteins.
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7 protocols using mouse enzyme linked immunosorbent assay kit
1
Quantifying IL-1β in BV2 Cells
2
Exorbital Lacrimal Gland Protein Analysis
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After euthanasia, the exorbital lacrimal glands of both eyes were dissected, snap frozen in liquid nitrogen, and stored at −70° C in a microcentrifuge tube. The frozen lacrimal tissues were weighed and cocktail protease inhibitor (1 mL 0.1 M phenylmethylsulfonyl fluoride, 50 µL aprotinin [1 mg/mL], 50 µL Triton X 100 and 50 mL phosphate buffer saline [PBS, pH 7.4] for volume) added to prepare a 10% w/v tissue homogenate. Each lacrimal gland tissue was homogenized for 30 to 90 seconds at 19,000 to 26,000 rotations per minute (rpm) in a 5 mL cryovial in a homogenizer in an ice bath. Following each run, the homogenizer probe was washed with 70% v/v isopropyl alcohol and PBS (pH 7.4) and wiped dry. All homogenized tissue samples were centrifuged at 10,000 rpm for 10 minutes at 4° C; the resulting supernatant was separated in microcentrifuge tubes, snap frozen in liquid nitrogen, and stored at −70° C. IL 1-β estimation in the supernatant was performed using mouse enzyme-linked immunosorbent assay kits (R&D Systems; catalog number: MLB00C) according to the manufacturer’s instructions.
3
Intestinal Cytokine and Immunoglobulin Profiling
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The faeces, duodenum, jejunum, ileum and colon, which were preserved at −80 °C, were thawed and the adipose tissue was removed. Pre-cooled sterile PBS solution was added according to a weight ratio of 1:9. The samples were then placed into a centrifuge tube together with cleaned and sterilised zirconia beads, broken down with a high-throughput crusher and then centrifuged at 4 °C for 2000× g for 10 min [18 (link)].
The supernatant was collected for assay measurement. Interleukin (IL)-1β, IL-6 and IL-17 concentrations were measured using mouse enzyme-linked immunosorbent assay kits according to the manufacturer’s protocol (R&D, Minneapolis, MN, USA). IgA and IgG concentrations were measured using mouse enzyme-linked immunosorbent assay kits according to the manufacturer’s protocol (Elabscience Biotechnology Co., Ltd., Wuhan, China). Protein was measured in intestinal tissue and faeces using the bicinchoninic acid assay (Beyotime Biotechnology, Shanghai, China).
The supernatant was collected for assay measurement. Interleukin (IL)-1β, IL-6 and IL-17 concentrations were measured using mouse enzyme-linked immunosorbent assay kits according to the manufacturer’s protocol (R&D, Minneapolis, MN, USA). IgA and IgG concentrations were measured using mouse enzyme-linked immunosorbent assay kits according to the manufacturer’s protocol (Elabscience Biotechnology Co., Ltd., Wuhan, China). Protein was measured in intestinal tissue and faeces using the bicinchoninic acid assay (Beyotime Biotechnology, Shanghai, China).
4
Cytokine Quantification of T Cell Subsets
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After culturing, conditioned medium was collected to examine cytokines. The cytokine levels were determined using mouse enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN, USA) for interferon (IFN)-γ, interleukin-2, and tumor necrosis factor (TNF)-α for CD4+ T cells, and IFN-γ for CD8+ T cells, respectively, using the Luminex 100 detection system (Luminex, Austin, TX, USA).
5
ChIP, ELISA, and MTT Assays for Transcription Factor
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ChIP assay was using a ChIP Assay Kit (Millipore, Massachusetts, USA). The quantity of DNA was analyzed by qPCR, gene-speci c primers of promoter used as Table S1.
Enzyme-linked immunosorbent assay (ELISA) Quantity of IL-1β in the supernatant of NC and shMEF2D stable BV2 cells were tested using mouse enzyme-linked immunosorbent assay kits (R&D Systems, Minnesota, USA) according to manufacturer procedures.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay NC and shMEF2D stable BV2 cells were seeded in 96-well plates (5 × 10 3 cells/well), then treated with LPS (500 ng/ml) for 24 and 48 h. 20 μl of MTT (5 mg/ml) (Millipore) were added to each well 4 hours before the very detection time. Then, after removing supernatant, 150 μl of dimethylsulfoxide (DMSO) (Millipore) was added to each well and mixed thoroughly for 10 min. The optical density was measured at 490 nm.
Enzyme-linked immunosorbent assay (ELISA) Quantity of IL-1β in the supernatant of NC and shMEF2D stable BV2 cells were tested using mouse enzyme-linked immunosorbent assay kits (R&D Systems, Minnesota, USA) according to manufacturer procedures.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay NC and shMEF2D stable BV2 cells were seeded in 96-well plates (5 × 10 3 cells/well), then treated with LPS (500 ng/ml) for 24 and 48 h. 20 μl of MTT (5 mg/ml) (Millipore) were added to each well 4 hours before the very detection time. Then, after removing supernatant, 150 μl of dimethylsulfoxide (DMSO) (Millipore) was added to each well and mixed thoroughly for 10 min. The optical density was measured at 490 nm.
6
Cytokine Levels Quantification in Mice
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The levels of IL-10, IL-6, and TNF-α were assessed using a mouse enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions.
7
PTFE Nanofibrous Membranes Influence on IL-6 Release
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The effect of the PTFE nanofibrous membranes on the amount released of the pro-inflammatory factor interleukin-6 (IL-6) was determined using the murine macrophage cell line RAW 264.7 which was obtained from the Korean cell line bank (KCLB, Korea). The RAW 264.7 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and antibiotic/antimycotic in a humidified atmosphere under 5% CO2 at 37 °C. For the cytokine immunoassay, the cells (2 × 104 cells per well) were seeded on the nanofibrous membranes in a 24 well tissue culture plate and cultured for 24 h at 37 °C. After that, 1 μg mL−1 of LPS was added to the cell culture plate to activate the cells for 24 h. The concentration of IL-6 was determined using a commercial mouse enzyme-linked immunosorbent assay kit (R&D Systems, USA). The kit was used according to the manufacture's specifications.
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