HaCaT cells were cultured in a 12-well plate for 24 h before the addition of fullerene or fullerenol (1 μM). After they received treatment for 1 h, cells were exposed to PM for another 24 h. The medium was collected for further measuring PGE2 levels using the PGE2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA), as per the manufacturer’s instructions. Three independent experiments were performed and values were calculated.
Pge2 enzyme immunoassay kit
Manufactured by Cayman Chemical
Sourced in United States, Italy
The PGE2 enzyme immunoassay kit is a laboratory tool used to quantify the concentration of prostaglandin E2 (PGE2) in samples. It utilizes an enzyme-linked immunosorbent assay (ELISA) principle to detect and measure PGE2 levels.
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Lab products found in correlation
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Anti cox 2 antibody, by Cayman Chemical (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Ns 398, by Beyotime (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Nitrate nitrite colorimetric assay kit, by Cayman Chemical (1 mentions)
Polytron homogenizer, by Thermo Fisher Scientific (1 mentions)
Elisa microplate reader, by PerkinElmer (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
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Luciferase assay substrate, by Promega (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Mts solution, by Promega (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Gemini Bio (1 mentions)
31 protocols using pge2 enzyme immunoassay kit
1
Fullerene Effects on PM-Induced PGE2
2
Mouse miR-214 Mimic Modulates COX-2 Signaling
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Dulbecco's modified Eagle's medium (DMEM), trypsin solution (EDTA), fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Gibco (Invitrogen, Grand Island, NY). UA was obtained from Sigma (St. Louis, MO). The mouse miR-214 mimic was provided by GenePharma Co., Ltd. (Shanghai, China). The COX-2 inhibitor NS-398 (catalog no. s1772) was purchased from Beyotime (Shanghai, China). The PGE2 enzyme immunoassay kit and the anti-COX-2 antibody were obtained from Cayman Chemicals (Ann Arbor, MI). The anti-BAX and GAPDH antibodies were provided by Proteintech (Rosemont, 90 USA). Anti-cleaved Caspase-3 antibody was purchased from Cell Signaling Technology (Danvers, MA).
3
Quantifying PGE2 in Cell Culture Media
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A PGE2 enzyme immunoassay kit (Cayman Chemical Co, Ann Arbor, MI) was used according to the manufacturer's instructions to quantify PGE2 concentrations in RIF-1 cells culture media. The 96 wells plate of the kit is coated with Goat Polyclonal Anti-Mouse IgG. This Competitive Enzyme Immunoassay is based on the competition between PGE2 present in the culture media and a PGE2-acetylcholinesterase conjugate called Tracer (provided by the kit) for a limited amount of PGE2 mouse monoclonal antibody (provided by the kit) in the well. The amount of the Tracer that is able to bind to the PGE2 monoclonal antibody and immobilized by the binding with the coated Anti-mouse IgG will be inversely proportional to the concentration of PGE2 of the culture media loaded in the well. The plate is washed to remove any unbound reagents and the Acetylcholinesterase substrate is added to each well. The product of this enzymatic reaction has a yellow color and absorbs at 410 nm. The intensity of this color, determined spectrophotometrically, is inversely proportional to the amount of PGE2 present in the culture media of each well.
The kit provides PGE2 of known concentration to prepare a standard curve (correlating 410 nm OD readings with PGE2 concentrations) used to calculate the unknown PGE2 levels in RIF-1 cells culture media.
The kit provides PGE2 of known concentration to prepare a standard curve (correlating 410 nm OD readings with PGE2 concentrations) used to calculate the unknown PGE2 levels in RIF-1 cells culture media.
4
PGE2 Production in HaCaT Cells
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HaCaT cells were cultured in 12-well culture plates. After reaching confluence, cells were treated with PMs for different time durations (as indicated). After treatment, medium was collected and stored at −80 °C until assays were performed. PGE2 production was assayed using the PGE2 enzyme immunoassay kit (Cayman), according to the manufacturer’s instructions.
5
UVB-Induced PGE2 Release Assay
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DHGA-D was treated to HaCaT cells plated in 6-well dishes at 80% confluency, 1 hr prior to UVB (0.05 J/cm2) irradiation, and then harvested 18 hrs later. The quantity of PGE2 released into the medium was measured by using a PGE2 enzyme immunoassay kit (Cayman Chemical Company, Ann Arbor, MI, USA).
6
Cell Line Immunoblotting Protocol
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IS was purchased from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin, and trypsin-EDTA solution were purchased from Gibco (Invitrogen, Grand Island, NY). Cyclin D1 mouse monoclonal antibody and cyclin A2 rabbit polyclonal antibody were from Abcam. COX-2 mouse monoclonal antibody was purchased from Cayman Chemicals (Ann Arbor, MI). Anti-GAPDH (ab9485) was provided by Cell Signaling Technology (Danvers, MA). The PGE2 enzyme immunoassay kit was from Cayman Chemicals (Ann Arbor, MI). COX-2 inhibitor, NS-398, was bought from Beyotime (Shanghai, China).
7
Measuring PGE2 Release from Caco-2 Cells
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PGE2 released by Caco-2 cells in the medium was measured by a PGE2 Enzyme Immunoassay Kit (Cayman Chemical Corporation, Milan, Italy) in accordance with the manufacturer’s instructions. Briefly, after treatment, cells were pelleted by centrifugation at 450× g for 5 min at 4 °C. Supernatants were diluted at 1:2.5 with assay buffer. A 100 µL sample, a 50 µL alkaline phosphatase PGE2 conjugate and a 50 µL monoclonal anti-PGE2 EIA antibody were, then, applied to a goat anti-mouse IgG-containing microtiter plate and incubated at room temperature for 2 h. After washing, 200 µL of p-nitrophenyl phosphate substrate solution was added and incubated at room temperature for 45 min. Finally, optical density at 405 nm was measured on a microplate reader (LTek, INNO, Seongnam, Republic of Korea). PGE2 concentrations in the samples were calculated from a PGE2 standard curve (25–2000 pg/mL) that was run in parallel.
8
Nitrite and PGE2 Measurement in LPS-Induced RAW 264.7 Cells
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RAW 264.7 cells were pre-treated with either NLEE or NLWE (1, 10, or 50 µg/ml) for 24 h and then induced with LPS (1 µg/ml) for 18 h. The supernatants was collected and the levels of nitrite and PGE2 were determined by a nitrate/nitrite Colorimetric Assay kit and a PGE2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA), in accordance with the manufacturer's protocol.
9
Quantification of PGE2 in Skin
10
Evaluating Anti-Inflammatory Potential of Plant Extracts
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HaCaT cell line (Keratinocyte cells) was cultured in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% heat inactivated fetal bovine serum (Sigma) and 100 µg/mL penicillin. Cells were cultured in a 5% CO2 atmosphere at 37°C. Cell viabilities were evaluated using MTT assay.[18 (link)] HaCaT cells (1 × 104/90 μL of DMEM medium) were seeded in 96-well plates. After overnight incubation, plant extract samples were treated for 72 h and after that 10 µL of MTT (5 mg/mL stock solution) was added into each well, incubated for 4 h at 5% CO2 atmosphere at 37°C. Medium was then removed and 150 μL of DMSO was added to dissolve Formazan crystals. Absorbance was measured at 540 nm using ELISA microplate reader (Perkin-Elmer, California, USA). Extracellular PGE2 releases were determined in HaCaT cell line using various concentrations of five most potent plant extracts as a 15-PGDH inhibitor. HaCaT cells were seeded (5 × 105 cells/well) into six-well culture plates in DMEM, supplemented with fetal bovine serum and antibiotics for overnight under 5% CO2, 37°C. Various concentrations of plant extract were added into individual well and media collected after 6 h for extracellular determination and cells were harvested for intracellular determination of PGE2. Concentrations of PGE2 were determined using PGE2 enzyme immunoassay kit (Cayman, MI, USA).
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