The secretion of TNF-α (cat. no. ml002859-1), IL-1β (cat. no. ml058228-1), IL-10 (cat. no. ml037888-1) and IL-18 (cat. no. ml063131-1) into the cell culture supernatant was analyzed using ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd.), according to the manufacturers' protocols. Briefly, the treated cells were harvested by centrifugation at 4˚C, 12,000 x g for 10 min. Then, the supernatant was collected and plated into ELISA microplates to measure the absorbance of each well at a wavelength of 450 nm using an automatic microplate reader (Syngene). Standard curves were drawn, and the concentrations of the inflammatory factors were expressed as pg/ml.
Automatic microplate reader
Manufactured by Syngene
Sourced in United States
The Automatic Microplate Reader is a laboratory instrument used to measure and analyze the optical properties of samples in microplate format. It provides accurate and reliable results for a variety of applications, including absorbance, fluorescence, and luminescence measurements.
Automatically generated - may contain errors
Lab products found in correlation
Elisa kit, by Shanghai Enzyme-linked (3 mentions)
Elisa kit, by Nanjing Jiancheng (2 mentions)
Elisa kit, by Beyotime (1 mentions)
Interleukin 6 (il 6), by Beyotime (1 mentions)
Tnf α, by Beyotime (1 mentions)
Mcp 1, by Beyotime (1 mentions)
Il 1β, by Beyotime (1 mentions)
Sbd f, by Merck Group (1 mentions)
Cobas 8000 modular analyzer series, by Roche (1 mentions)
Corresponding kit, by Nanjing Jiancheng (1 mentions)
9 protocols using automatic microplate reader
1
Measuring Inflammatory Cytokine Secretion
2
VEGF Quantification via ELISA
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An ELISA kit (Nanjing Jiancheng Bioengineering Institute; cat. no. H044-1) was used to analyze the levels of VEGF according to the manufacturer's protocol. Each group was quantified using an Automatic Microplate Reader (Syngene).
3
Serum Cytokine Levels in Septic Mice
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TNF-α (PT512), IL-6 (cat. no. PI326) and IL-1β (PI301) levels in the serum of septic mice were detected using respective ELISA kits according to the manufacturer's instructions (Beyotime Institute of Biotechnology). In brief, 2 ml blood was collected from mice at room temperature and centrifuged at 4°C for 5 min at 3,000 × g. The absorbance of the serum sample was measured at 450 nm using an Automatic Microplate reader (Syngene).
4
Quantifying LDH Release in Cell Supernatant
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Cell supernatant was collected and centrifuged at 700 × g at 4°C for 10 min. Then centrifugation was performed at 9,000 × g at 4°C for another 15 min and the supernatant collected. According to the manufacturer's instructions, an ELISA kit (cat. no. A020-1-2; Nanjing Jiancheng Bioengineering Institute) was used to analyze the level of lactate dehydrogenase (LDH) release in the supernatant. The samples of each group were analyzed using an Automatic Microplate Reader (Syngene). All the experiments in this study were repeated three times.
5
Quantification of Inflammatory Markers
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Levels of IL-1β (#PI305; Beyotime Biotechnology, Shanghai, China), IL-6 (#PI330; Beyotime Biotechnology), TNF-α (#PT518; Beyotime Biotechnology) and MCP-1 (#PC130; Beyotime Biotechnology) in serum with commercially available standard sandwich enzyme-linked kits in accordance with the manufacturer's instructions. The samples of each group were quantified by the Automatic Microplate Reader (Syngene, Frederick, MD, USA). Each sample was measured in triplicate.
6
Plasma Glucose, Insulin, and Homocysteine Analysis
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2 ml blood from the antecubital vein were immediately placed on ice and separated into plasma and cells within 30 min, then stored at – 80 °C until analysis. Plasma glucose measurements were performed by glucose oxidase method on the Cobas 8000 Modular Analyzer Series (Roche, Mannheim). Fasting plasma insulin (FPI) was determined by enzyme-linked immunosorbent assay (ELISA) with the Insulin ELISA 10-1113-10 kit (Mercodia, Sweden, with intraassay CV of 5.91% and between-assay CV of 7.15%). The absorbance was measured at 450 nm using the Automatic Microplate Reader (Syngene, BioTek, USA). Homeostasis model of insulin resistance (HOMA-IR) was used to evaluate insulin resistance. HOMA-IR was calculated as fasting glucose × fasting insulin/22.5. HOMA-beta cell function (HOMA-f) was calculated as 20 × fasting insulin/(fasting glucose − 3.5).
Plasma total homocysteine (tHcy: the sum of all Hcy forms) was measured by high-performance liquid chromatography (HPLC, Waters 1525, USA) with fluorescence detection, applying 7-fluorobenzo-2oxa-1,3-diazole-4-sulfonate (SBD-F, Sigma, USA) as derivating agent and tris(2-carboxyethyl)phosphine (TCEP, Sigma, USA) as reducing agent27 (link). The intraassay CV was 3.60%, and between-assay CV was 4.49%.
Plasma total homocysteine (tHcy: the sum of all Hcy forms) was measured by high-performance liquid chromatography (HPLC, Waters 1525, USA) with fluorescence detection, applying 7-fluorobenzo-2oxa-1,3-diazole-4-sulfonate (SBD-F, Sigma, USA) as derivating agent and tris(2-carboxyethyl)phosphine (TCEP, Sigma, USA) as reducing agent27 (link). The intraassay CV was 3.60%, and between-assay CV was 4.49%.
7
Quantifying Inflammatory Cytokine Levels
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After the end of the reoxygenation stage, the culture supernatants were collected. The concentration of inflammatory cytokines TNF-α (cat. no. F16960), IL-6 (cat. no. F15870) and IL-1β (cat. no. F15810), was determined using ELISA according to the manufacturer's protocol (Shanghai Xitang Biotechnology Co., Ltd.). Briefly, the treated cells were harvested by centrifugation at 4˚C, 12,000 x g for 10 min. The supernatant was then collected and plated into ELISA microplates to measure the absorbance of each well at a wavelength of 450 nm using an automatic microplate reader (Syngene).
8
Oxidative Stress Biomarker Analysis
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Following the manufacturer’s instructions, the corresponding kits (all from Nanjing Jiancheng Bioengineering Institute) were used to analyze the levels of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione (GSH) released in the supernatant. The samples of each group were quantified with an automatic microplate reader (Syngene).
9
Quantification of Inflammatory Cytokines
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According to the manufacturer's protocols, corresponding ELISA kits (Nanjing Jiancheng Bioengineering Institute) were used to analyze the levels of IL-6 (IL-6 Assay Kit, cat. no. H007-1-1), TNF-α (TNF-α Assay Kit, cat. no. H052-1) and IL-1β (IL-1β Assay Kit, cat. no. H002) in the cell culture supernatant. The samples in each group were analyzed using an Automatic Microplate Reader (Syngene).
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