Cells (2 × 105 per well) were stimulated, or not, as described above, for 48 h. Briefly, cells supernatant were collected, centrifuged, and then kept at − 20 °C for later use. Interleukin 8 (IL-8) and PGE2 were dosed using IL-8 EIA KIT-monoclonal (Cayman Chemicals) and PGE2 EIA KIT-Monoclonal (Cayman Chemicals), respectively, following the protocol described by the manufacturer. The result was measured in a spectrophotometer set to 420 nm. The result was given as pg/μL, relating the absorbance of the samples to a standard curve present in the kit.
Pge2 eia kit monoclonal
Manufactured by Cayman Chemical
Sourced in United States
The PGE2 EIA Kit-Monoclonal is a laboratory equipment product designed for the quantitative determination of Prostaglandin E2 (PGE2) levels in a variety of sample types. The kit utilizes a competitive enzyme immunoassay (EIA) technique with a monoclonal antibody specific to PGE2.
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C18 spe column, by Waters Corporation (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Creatinine colorimetric fluorometric assay kit, by Abcam (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Quant it picogreen dsdna assay kit, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Dutscher (1 mentions)
Collagenase crude type 1 a, by Merck Group (1 mentions)
Protanal lf 10 60 ft, by FMC Biopolymer (1 mentions)
Fluorescein isothiocyanate fitc dextran, by Merck Group (1 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Calcium chloride, by Merck Group (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
Mm300, by Qiagen (1 mentions)
Bcatm protein assay kit, by Thermo Fisher Scientific (1 mentions)
Spectramax 250, by Molecular Devices (1 mentions)
7 protocols using pge2 eia kit monoclonal
1
Quantifying IL-8 and PGE2 Secretion
2
Quantifying PGE2 Levels in Biological Samples
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PGE2 levels were measured by commercially available enzyme immunoassay (PGE2 EIA Kit-Monoclonal; Cayman Chemical, Tallin, Estonia). Samples were sonicated in 400mL homogenization buffer (0.1M phosphate buffer, pH=7.4, 1mM EDTA, and 10mM indomethacin), purified in 4 volumes ethanol for 5 minutes at 4°C, centrifuged at 3000 g for 10 minutes, and acidified with glacial extracted using SPE (C-18) acetic acid (pH=3.5). PGE2 was extracted using SPE (C-18) columns (Waters, MA) rinsed with methanol and water. After sample′s application, columns were washed with water and hexane and PGE2 was eluted with ethyl acetate. Samples were evaporated to dryness under nitrogen and resuspended in enzyme immunoassay buffer. PGE2 levels were measured in a 96-well plate and read at 405nm following the manufacturer’s instructions (Synergy 2; BioTek Instruments). The sensitivity of the assay for PGE2 was 15 pg/mL; intra- and interassay coefficients of variation were 6.6% and 15.5%, respectively.
3
Urinary Biomarkers in Renal Dysfunction
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At the end of the 16-week treatment, mice were placed in mouse metabolic cages for 24 h urine collection. Urine samples were centrifuged and stored at −80 °C. Urinary albumin and noradrenaline were measured with enzyme-linked immunosorbent assay (ELISA) kits (Mouse Albumin ELISA, #1011, Exocell, Philadelphia, PA, USA; Norepinephrine ELISA Kit, #E4360, Biovision, Milpitas, CA, USA). Urinary kidney injury molecule-1 (KIM-1) as a marker of renal injury was quantified using a mouse kim-1 Elisa kit (CL0880, Cell applications Inc, CA, USA) according to the manufacturer’s protocol. Plasma creatinine concentrations were assayed using the Creatinine Colorimetric/Fluorometric Assay Kit (K625, Biovision, Milpitas, CA, USA). Endogenous creatinine clearance is a sensitive and accurate method for assessing glomerular filtration rate (GFR). Plasma and urine creatinine levels were determined using the above-mentioned assay kit (K625, Biovision) and calculation of creatinine clearance: GFR = U[Cr] × Volume]/P[Cr] × [Time] [25 (link)]. Since the half-life of PGI2 was short, 6-keto-PGFla, its stable and inactive metabolite, was measured. A PGE2 EIA Kit-Monoclonal (#514010, Cayman Chemical Company, MI, USA) and a 6-keto-PGFla EIA Kit (#515211, Cayman Chemical Company, MI, USA) were used according to the manufacturer’s instructions.
4
Quantitative PGE2 Immunoassay for cPLA2α Activity
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A sensitive, quantitative enzyme immunoassay specific for the metabolite PGE2 was used as a proxy to assess cPLA2α activity. Supernatant from cells were spun at 1500 rpm for 10 min at 4 °C and stored at −80 °C. After thawing, supernatants were spun at 2000 rpm for 5 min at 4 °C, diluted 1:50 in DMEM, and analyzed in duplicate using a 96-well PGE2 EIA Kit Monoclonal (Cayman Chemical Company, Ann Arbor, MI, USA). The analysis was performed using the four parameter logistic curve function in the MyAssays Prostaglandin E2 Monoclonal online analysis tool [57 ]. A two-tailed Student’s t-test was used to evaluate the statistical significance (p < 0.05).
5
PGE2 Production Quantification in DCs
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The production of PGE2 was assessed by ELISA (PGE2 EIA Kit-Monoclonal; Cayman Chemicals, Ann Arbor, MI), in supernatants obtained from cell cultures, 24 hours after liposome capture by DCs. Limit of detection: 80% B/B0: 15 pg/ml. Sensitivity: 50% B/B0: 50 pg/ml. Results were expressed as pg of PGE2/106 cells.
6
Hydrogel-based cell culture platform
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Sodium alginate (Protanal™LF10/60FT) and hydroxypropyl methylcellulose (HPMC) (Methocel™E4M) were purchased, respectively, from FMC Biopolymer and Colorcon-Dow chemical (Bougival, France). Glycidoxypropyltrimethoxysilane (GPTMS) was obtained from Acros (Geel, Belgium). Hank's balanced sodium salt (HBSS), Dulbecco's modified eagle medium (DMEM) high glucose (4.5 g/L), phosphate-buffered salt (PBS) without Calcium chloride and magnesium chloride, penicillin/streptomycin, and trypsin/EDTA (0.05%/0.53 mM) were obtained from Invitrogen (Paisley, UK). Calcium chloride, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), olive oil, fluorescein isothiocyanate- (FITC-) dextrans, collagenase crude type I A, trypan blue, sodium citrate, and trichloroacetic acid (TCA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal calf serum (FCS) was purchased from Dominique Dutscher (Brumath, France). Live/Dead Viability/Cytotoxicity kit and Quant-iT PicoGreen dsDNA assay kit were, respectively, obtained from Molecular Probes (Leiden, The Netherlands) and Thermo Fisher (Waltham, MA, USA). PGE2 EIA Kit-Monoclonal and Human HGF Duo Set ELISA were purchased from Cayman Chemical and R&D Systems, respectively.
7
Quantification of Bladder PGE2 Levels
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After measuring the weight of mucosal tissue of the bladder, homogenization (300Hz/s, 3 min) was conducted with a mixer mill (MM300; Qiagen). The protein concentration in the supernatant was measured with the BCATM protein assay kit (Thermo Fisher Scientific). The urine and retrieved intravesical irrigation samples were centrifuged at 3000g for 10min at 4°C.
The supernatant was separated into aliquots in 96 well plates and preserved in a freezer at -80°C .
The level of PGE2 was measured by enzyme-linked immunosorbent assay (ELISA). To measure the amount of PGE2, a PGE2 EIA Kit-Monoclonal (Cayman Chemical) was used according to the methods described in the manual. The concentration was determined following absorbance measurement with a microreader (SpectraMax 250; Molecular Devices Japan). The total tissue PGE2 (pg/ml) was normalized by the weight of tissue and the ratio of PGE2/ the weight of tissue used as a normalized tissue PGE2 level. The total urinary PGE2 level (pg/ml) was normalized by the urinary creatinine and the ratio of PGE2/creatinine used as a normalized urinary PGE2 level.
The supernatant was separated into aliquots in 96 well plates and preserved in a freezer at -80°C .
The level of PGE2 was measured by enzyme-linked immunosorbent assay (ELISA). To measure the amount of PGE2, a PGE2 EIA Kit-Monoclonal (Cayman Chemical) was used according to the methods described in the manual. The concentration was determined following absorbance measurement with a microreader (SpectraMax 250; Molecular Devices Japan). The total tissue PGE2 (pg/ml) was normalized by the weight of tissue and the ratio of PGE2/ the weight of tissue used as a normalized tissue PGE2 level. The total urinary PGE2 level (pg/ml) was normalized by the urinary creatinine and the ratio of PGE2/creatinine used as a normalized urinary PGE2 level.
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