The evaluation of cytokine release from LPS-stimulated neutrophils was assessed following a previously described protocol [56 (link)]. Thus, neutrophils were incubated at 37 °C in a 96-well plate in RPMI 1640 medium (prepared as described in Section 4.9.2 .), with/without honokiol or magnolol (12.5, 25 and 50 µM) added 30 min prior to neutrophils’ stimulation with LPS 100 ng/mL. After 24 h, the plates were centrifuged, the supernatants were collected and the cytokines’ release was determined by ELISA assay kits following the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA) using a Synergy 4 BioTek microplate reader (Winooski, VT, USA). The effect on cytokine production was determined through the percentage of released cytokines relative to the control without the test compounds. Urolithin A (a substance with known anti-inflammatory properties [57 (link),58 (link)], previously acquired by isolation (>95% HPLC-DAD purity) in the Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw, Warsaw, Poland) at concentrations of 12.5, 25 and 50 µM was used as a positive control.
Elisa assay kit
Manufactured by BD
Sourced in United States
The ELISA assay kit is a laboratory equipment designed for enzyme-linked immunosorbent assay (ELISA) analysis. It provides the necessary components and protocols to perform quantitative or qualitative detection and measurement of specific analytes in a sample. The kit includes essential reagents, microplates, and instructions for conducting the ELISA procedure.
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Lab products found in correlation
Rpmi 1640 media, by Thermo Fisher Scientific (1 mentions)
Human thp 1 monocytes, by American Type Culture Collection (1 mentions)
Lps from e coli o55 b5, by Merck Group (1 mentions)
Escherichia coli 055 b5, by Merck Group (1 mentions)
Il 1β, by BD (1 mentions)
Fluoroskan ascent fl, by Thermo Fisher Scientific (1 mentions)
Insulin, by Cayman Chemical (1 mentions)
Reactive strips, by Johnson & Johnson (1 mentions)
Tnf α and il 6, by BD (1 mentions)
7 protocols using elisa assay kit
1
Cytokine Release from LPS-Stimulated Neutrophils
2
Cytotoxicity and Anti-inflammatory Evaluation of Extracts
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Human THP-1 monocytes (ATCC, Manassas, VA, USA) were maintained and cultured in supplemented RPMI 1640 media (Gibco, Paisley, UK) until confluence to perform the cytotoxicity and anti-inflammatory assays as described by Villalva et al. [24 (link)]. For differentiation of THP-1 monocytes to macrophages, cells were seeded in 24-well plates (5 × 105 cells/mL) with 100 ng/mL of PMA for 48 h. The cytotoxicity of the extracts was tested (10, 20 and 50 using µg/mL) on differentiated macrophages using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium Bromide (MTT). For anti-inflammatory assays, macrophages were incubated with or in absence of 0.05 µg/mL of bacterial lipopolysaccharide (LPS from E. coli O55:B5, Sigma-Aldrich, Madrid, Spain) in the presence of a non-cytotoxic extract concentration for 24 h. The secretion of the pro-inflammatory cytokines, TNF-α, IL-1β and IL-6, was measured in the cell’s supernatants using ELISA assay kits (BD Biosciences, Aalst, Belgium), according to the manufacturer’s instructions. Results were expressed as mean of three determinations ± standard deviation.
3
ELISA-based Cytokine Profiling
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For cytokine assays, supernatants were collected at different time points post BCG infection and used to measure IL-10, TNF-α, IFN-γ, and IL-6 concentrations using ELISA assay kits as per manufacturer recommendations (BD Biosciences).
4
Microglia Isolation and Activation Protocol
5
Lactobacillus Strains Attenuate Inflammatory Mediators
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The protection effect of Lactobacillus strains against inflammation mediators (IL-8 and NO) of Caco-2 cells stimulated with 1 ng/mL IL-1β (Pharmingen, NJ, USA) or 0.1 µg/mL LPS of E. coli 0111:B4 for 24 h was evaluated measuring the IL-8 levels by ELISA assay kit (Pharmingen, NJ, USA) under basal conditions, with stimulated or without the Lactobacillus strains stimulus (ratio 1:1) after 24 h. The NO production levels were quantified by the Griess reaction in accordance with methods described by Archer, et al. [24 (link)]. The accumulation of nitrites, as a measure of the secretion of NO, was determined in the cell culture supernatant reading the absorbance at 570 nm using a microplate reader Fluoroskan Ascent FL (Thermo Fisher Scientific, Waltham, MA, USA). The culture medium alone was employed as the blank control.
6
Quantification of Human IL-1β by ELISA
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Cells were cultured and treated as described above. Supernatants were harvested and frozen for human IL-1β quantification using an ELISA assay kit (BD Biosciences) according to the manufacturer's instructions [36] (link).
7
Dietary Intervention in Obesity-Induced Metabolic Changes
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All experimental procedures were performed in accordance with the Brazilian National Law and were approved by the by Ethics Committee of the Institute of Biomedical Sciences (ICB), University of São Paulo (USP) (number 195/11). Four-week-old C57BL/6 male mice were divided into two groups: (1) control group, which was fed a regular chow (70% carbohydrate, 20% protein, and 10% fat), with a caloric content of 3.8 kcal/g; (2) obese group, which was fed a high-fat diet (26% carbohydrate, 15% protein, and 59% fat) (PragSoluções, Brazil), with a caloric content of 5.4 kcal/g. The animals were maintained on a 12:12-hour light-dark cycle in a temperature-controlled environment (22 ± 2°C) with free access to food and tap water.
After 16 weeks subjected to a high-fat diet, the animals were fasted for 6 hours, weighed, and anesthetized with intraperitoneal injection of ketamine (150 mg/kg) and xylazine (7.5 mg/kg). Periepididymal and retroperitoneal fat deposits were excised and weighed. Blood samples were collected by cardiac puncture and the serum was used for biochemical assays. Triglycerides and total, HDL, and LDL cholesterol levels were measured by colorimetric kit assays (Labtest, Brazil). Glucose level was assessed by reactive strips (Johnson & Johnson, USA). Insulin (Cayman Chemical, USA) and TNF-α and IL-6 (BD Biosciences, USA) levels were assessed by ELISA assay kit.
After 16 weeks subjected to a high-fat diet, the animals were fasted for 6 hours, weighed, and anesthetized with intraperitoneal injection of ketamine (150 mg/kg) and xylazine (7.5 mg/kg). Periepididymal and retroperitoneal fat deposits were excised and weighed. Blood samples were collected by cardiac puncture and the serum was used for biochemical assays. Triglycerides and total, HDL, and LDL cholesterol levels were measured by colorimetric kit assays (Labtest, Brazil). Glucose level was assessed by reactive strips (Johnson & Johnson, USA). Insulin (Cayman Chemical, USA) and TNF-α and IL-6 (BD Biosciences, USA) levels were assessed by ELISA assay kit.
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