The ex vivo generation of eicosanoids and cytokines in the small intestine tissue section was detected using a modified method previously described [46 (link)]. Immediately after removal, a 2 cm intestinal section was minced, and washed in cold Tyrode buffer and resuspended in 37°C freshly oxygenated Tyrode buffer (Sigma-Aldrich). After incubation at 37°C for 20 min, the supernatants were collected. Levels of LTB4 and PGE2 were determined using enzyme immunoassay kits (Cayman Chemical). Analysis of cytokines present in the same intestinal supernatants was determined by using a Milliplex MAP immunoassay kit (Millipore) that was analyzed on a Milliplex Analyzer (Millipore). Supernatants generated for eicosanoids and cytokines were also used to determine peroxidase activity by measuring oxidation of 3,3’,5,5’-tetramethylbenzidine (TMB) as described previously[11 (link)]. Protein content was determined using the bicinchoninic acid (Pierce) assay adapted for use with microtiter plates. All eicosanoid and cytokines concentrations were standardized per mg protein per 20 min.
Tyrode buffer
Manufactured by Merck Group
Sourced in United States
Tyrode buffer is a saline solution commonly used in laboratory settings. It is composed of various salts and is designed to maintain the physiological pH and osmolarity of biological samples. The core function of Tyrode buffer is to provide a balanced ionic environment for the preservation and study of cells, tissues, or other biological materials.
Automatically generated - may contain errors
Lab products found in correlation
Enzyme immunoassay kit, by Cayman Chemical (2 mentions)
Milliplex analyzer, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
96 well cell culture plate, by Corning (2 mentions)
Victor plate reader, by PerkinElmer (2 mentions)
4 methylumbelliferyl n acetyl β d glucosaminide, by Merck Group (2 mentions)
Milliplex map immunoassay kit, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 culture medium, by Lonza (1 mentions)
6 protocols using tyrode buffer
1
Ex vivo intestinal eicosanoid and cytokine analysis
2
Ex vivo Intestinal Eicosanoid and Cytokine Profiling
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Mid-jejunal tissue ex vivo generation of eicosanoids and cytokines was determined by using an adapted procedure previously described (49 (link)). Briefly, immediately after collection, 2 cm mid-jejunum sections were minced, washed in pre-chilled Tyrode buffer (Sigma-Aldrich), and resuspended in 37°C freshly oxygenated Tyrode buffer. After 20 min incubation at 37°C, the supernatants and tissues were collected and stored at -80°C until assayed. The tissue protein content was detected by utilizing the bicinchoninic acid assay (Pierce, cat. # 23225) modified for use with microtiter plates. Bovine Serum Albumin (BSA) (Fisher Scientific, cat. # BP9703100) was used as a standard. The same intestinal tissue supernatants were used to determine concentrations of leukotriene B4 (LTB4) and prostaglandins E2 (PGE2) utilizing enzyme immunoassay kits (Cayman Chemical, cat. # 520111 and #500141, respectively). Supernatants generated for eicosanoids were also used to detect cytokine levels by using Milliplex MAP immunoassay kits (Millipore, custom kits) that were analyzed on a Milliplex Analyzer (Millipore). The concentrations of eicosanoid and cytokines were standardized per mg intestinal tissue protein per 20 min incubation period.
3
Rat Basophil Leukemia Cell Degranulation
4
Rat Basophil Leukemia Cell Degranulation
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Rat basophil leukemia (RBL) cells were grown in 96-well cell-culture plates (Costar, Corning, Tewksbury, Mass) for 20 hours at 37°C and 5% CO2. Aliquots of 5 μL of a serum pool from sensitized mice were added and the cells were incubated at 37°C, 5% CO2 for 2 hours and washed 2 times with Tyrode buffer (Sigma-Aldrich). Degranulation of RBL cells was induced by adding 100 μL of rCyp c 1 (0.3 μg/mL) in Tyrode buffer and incubation for 30 minutes at 37°C, 5% CO2. In the inhibition experiments, rCyp c 1 (0.3 μg/mL) was added together with 10% vol/vol mCyp c 1–specific heat-inactivated rabbit or mouse antiserum or control antiserum or buffer. Beta-hexosaminidase in culture supernatants was detected with 0.16 mmol 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide (Sigma-Aldrich) and fluorescence was measured at excitation and emission wavelengths of 360 to 465 nm λex-λem using a microplate reader (VICTOR Plate Reader; Perkin Elmer). Results are reported as percentages of total β-hexosaminidase released after cell lysis by addition of 10% Triton X-100 (Merck Millipore, Darmstadt, Germany). All measurements were performed in triplicates and standard deviations are given for triplicate determinations.
5
Coronary Artery Disease Platelet Isolation
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All experiments with human subjects were approved by the ethic committee at Shiraz University of Medical Sciences. 60 subjects including patients with acute coronary syndrome signed a consent form for participating in this study. All subjects submitted to coronary angiography at Shiraz University of Medical Sciences Hospital were divided into three groups by the specialist. Among patients (60 cases), 20 cases did not have any CAD (control group), 20 patients were diagnosed with CT group, and the other 20 possessed less than 70% Coronary Stenosis (CS group). Venous blood was taken from subjects in sterile conditions with EDTA potassium salt as an anticoagulant. Platelets were isolated from patients’ blood by centrifuging (190×g) of whole blood for 15 min; and then centrifuging (2500×g) for 15 min. The pellet of platelet was resuspended and kept in Tyrode buffer (Sigma, USA) at 22°C until doing experiments. Platelets were counted using a Goryaev chamber. The plasma of samples was stored at −70°C for measuring NO concentration.
6
Histamine Release in Canine Mast Cell Tumors
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Two canine MCT cell lines, C2 and BR, were maintained in C10 media (RPMI 1640 culture medium [Lonza Group Ltd] supplemented with heat-inactivated 10% fetal bovine serum [Peak Serum Inc] and a solution mixture [Cellgro] of 1X minimum essential medium, 2mM l-glutamine, 1mM sodium pyruvate, 1X nonessential amino acid solution, and 1X antimicrobial [bacteria and fungi] solution) and placed in a humidified incubator with 5% CO 2 at 37 °C. Cell lines were confirmed to be of canine origin by use of a multispecies multiplex PCR assay and authenticated with short-tandem repeated analysis as described. 12 The 2 X 10 6 C2 or BR cells were placed in 500 mL of Tyrode buffer (Sigma-Aldrich) and incubated for 1 hour at 37 °C in the presence of 1, 100, or 1,000 ng of morphine/mL in duplicate. Mastoparan (Cayman Chemical; 25mM) served as the positive control, and Tyrode buffer alone (untreated) served as the negative control. After incubation, the reactions were stopped with 15-minute incubation on ice, after which the supernatants were removed and stored at -80 °C until analysis. Samples were thawed, and histamine concentrations were measured in duplicate by use of an ELISA (Immunotech). The assay's sensitivity was 0.05 ng/mL and had been validated for histamine determination in dogs. [13] [14] [15]
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