At the time of inclusion, blood was collected in Sodium Citrate vacutainers at the outpatient clinic prior to surgery from a subpopulation of 35 randomly selected CEA patients. Blood was immediately centrifuged at 1850 G and subsequently isolated for LTB4 plasma measurements. To assess the comparison between plaque and plasma levels, LTB4 plasma levels were quantified using ELISA (LTB4 Parameter Assay Kit, R&D Systems Inc., Minneapolis, USA) and expressed as pg/ml of plasma. To validate the occurrence of ex vivo LTB4 formation after whole blood collection, a pilot validation study was performed with EDTA plasma (n = 4) and Sodium Citrate plasma (n = 4) preincubated with a 5-LOX inhibitor (100 µM Zileuton, Sigma Aldrich) or PBS (control) immediately after collection for 30 minutes. Plasma was collected and LTB4 levels were measured using ELISA (LTB4 Parameter Assay Kit, R&D Systems Inc., Minneapolis, USA) and expressed as pg/ml of plasma.
Ltb4 parameter assay kit
Manufactured by R&D Systems
Sourced in United States
The LTB4 Parameter Assay Kit is a laboratory equipment product designed for the quantitative measurement of Leukotriene B4 (LTB4) levels in biological samples. It provides a reliable and sensitive method for the detection and analysis of this important lipid mediator.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Facsaria 3, by BD (1 mentions)
Zileuton, by Merck Group (1 mentions)
Mouse tnf alpha elisa kit, by Abcam (1 mentions)
Lipid peroxidation mda assay kit, by Abcam (1 mentions)
Mouse mcp1 elisa kit, by Abcam (1 mentions)
Amplex red h2o2 assay kit, by Thermo Fisher Scientific (1 mentions)
Creatinine assay kit, by Abcam (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
8 protocols using ltb4 parameter assay kit
1
Quantifying LTB4 Levels in CEA Patients
2
Quantifying LTB4 in Embryonic Hematopoietic and Endothelial Cells
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HSPC (CD31+CD45−Kit+) and EC(CD31+CD45−Kit+) populations from E11.5 Ctr or cKO AGM were sorted by FACS Aria III (BD Biosciences) into 7% BSA in FACS buffer. Cells were precipitated by centrifugation at 2500 RPM, 5 min, 4 °C. Cells were lysed in PBS(-) by freeze-and-thaw for three times using liquid nitrogen and 37 °C water bath. Cells were then stored at −80 °C prior to ELISA. ECs from two embryos of the same genotype and early HSPCs from five embryos of the same genotype were pooled and the level of LTB4 was measured using LTB4 Parameter Assay kit (R&D KGE006B) according to manufacturer’s instructions.
3
Kidney Injury Biomarkers Quantification
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Urinary H2O2 and NGAL levels were measured in the resulting urine samples with the Amplex Red H2O2 assay kit (A12214, Invitrogen, Carlsbad, CA, USA) and Mouse Lipocalin-2/NGAL ELISA Kit (ab119601, Abcam, Cambridge, UK). Serum creatinine, MCP-1, TNFα, and LTB4 levels were measured with a creatinine assay kit (ab65340, Abcam, Cambridge, UK), Mouse MCP1 ELISA Kit (ab100721, Abcam), Mouse TNF alpha ELISA Kit (ab46105, Abcam), and LTB4 Parameter Assay Kit (KGE006B; R&D Systems, Minneapolis, MN, USA), respectively. Kidney malondialdehyde (MDA) levels were measured by a Lipid Peroxidation (MDA) Assay Kit (ab118970, Abcam).
4
Quantification of LTB4 in Atherosclerotic Lesions
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Adjacent segments of the culprit lesion were used for protein isolation as described previously [14] (link). In short, plaque segments were snap-frozen in liquid nitrogen and stored at −80°C until further use. Protein extraction was performed according to a standardized protocol using Tris buffer. Total protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific, Etten-Leur, The Netherlands). Expression levels of LTB4 within the atherosclerotic specimen were quantified using ELISA (LTB4 Parameter Assay Kit, R&D Systems Inc., Minneapolis, USA). LTB4 levels were adjusted for total protein concentration within the atherosclerotic specimen and expressed as pg/mg of total protein. For validation purposes, uncorrected LTB4 data is also depicted and expressed as pg/ul sample. Expression of a selection of proteins involved upstream and downstream of the ALOX5 pathway was analyzed using Western Blot and quantified as intensity per square millimeter. These were BLT1, ALOX5 and PKC.
5
Cytokine and Leukotriene B4 Profiling
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Plasma levels of tumor necrosis factor-α (TNF-α), IL-1β, IL-6, IL-8, IL-10, and GRO-α were measured with a Luminex bead assay using the Human XL Cytokine Discovery Panel (LKTM014, R&D Systems). Plasma LTB4 levels were measured by enzyme-linked immunosorbent (ELISA) assay using the LTB4 Parameter Assay Kit (KGE006B, R&D Systems). Each sample was assayed in duplicate.
6
Neutrophil Stimulation and LTB4 Secretion
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Neutrophils were resuspended in complete RPMI either untreated or stimulated with 2 × 104 zymosan particles for 15 and 30 min. Alternatively, neutrophils were stimulated with a mixture of phorbol myristate acetate (PMA) (100 ng/mL) and ionomycin (1 μg/mL) or thapsigargin (1 μM) for 30 min (all purchased from Sigma). Supernatants were recovered and the secretion of LTB4 was measured using a LTB4 parameter assay kit (R&D Systems).
7
Quantifying LTB4 and Lipoxygenase
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LTB4 concentrations in BAL fluid from mice and cell culture supernatants were determined using the LTB4 Parameter Assay Kit (R&D Systems) according to the manufacturer’s instructions. Lipoxygenase activity was determined in cell culture supernatants using the Lipoxygenase Assay Kit (Abcam) according to the manufacturer’s instructions.
8
Determination of Inflammatory Mediators in SU/Hy Rat Model
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The SU/Hy rat model was established according to the details described previously [15] . At the end of a 6-week model establishment, 4.5 mg•kg -1 of BUR1 or the vehicle control 0.5% carboxymethyl cellulose was administrated daily via the intragastric (i.g.) route for three more weeks before mice underwent cardiopulmonary phenotyping and were killed.
Quantification of PGE 2 and LTB 4 in the rat plasma Blood samples were collected from rats in the SU/Hy model assay. Prostaglandin E 2 (PGE 2 ) and LTB 4 levels in the plasma were measured using the Prostaglandin E2 high sensitivity ELISA Kit (Abcam, Cambridge, UK) and the LTB 4 Parameter Assay Kit (R&D Systems, Minneapolis, MN, USA) with a competitive ELISA method according to the manufacturers' instructions.
Additional information about reporter line generation, microarrays, endothelial cell differentiation from hESCs, CRISPR/Cas9-based mutation generation, immunostaining, MCT, SU/Hy animal models and other experiments can be found in the supplemental material.
Quantification of PGE 2 and LTB 4 in the rat plasma Blood samples were collected from rats in the SU/Hy model assay. Prostaglandin E 2 (PGE 2 ) and LTB 4 levels in the plasma were measured using the Prostaglandin E2 high sensitivity ELISA Kit (Abcam, Cambridge, UK) and the LTB 4 Parameter Assay Kit (R&D Systems, Minneapolis, MN, USA) with a competitive ELISA method according to the manufacturers' instructions.
Additional information about reporter line generation, microarrays, endothelial cell differentiation from hESCs, CRISPR/Cas9-based mutation generation, immunostaining, MCT, SU/Hy animal models and other experiments can be found in the supplemental material.
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