About 0.3 g of each jejunal and ileal mucosa sample was homogenized at a ratio of 1:9 (weight/volume) with ice-cold 154 mmol/L sterile sodium chloride solution using a PRO-PK-02200D homogenizer (Pro Scientific, Inc., Monroe, CT). The homogenate was centrifuged at 3,500 × g for 10 min at 4°C to acquire supernatant, and it was immediately frozen at −20°C for further analysis. Concentrations of malondiadehyde (MDA ) and glutathione (GSH ), and superoxide dismutase (SOD ), glutathione peroxidase (GSH-Px ), and catalase (CAT ) were determined in accordance with the manufacturer's instructions using available commercial kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, Jiangsu Province, P. R. China). The results were normalized against total protein concentration in each sample for intersample comparison. Total protein content of each mucosal sample was determined using a Coomassie brilliant blue protein assay kit (Nanjing Jiancheng Institute of Bioengineering, Nanjing, Jiangsu Province, P. R. China) using bovine serum albumin as the standard.
Pro pk 02200d homogenizer
Manufactured by PRO Scientific
The PRO-PK-02200D is a laboratory homogenizer designed for efficient sample preparation. It features a high-speed motor and a variety of interchangeable probes to accommodate different sample volumes and viscosities. The homogenizer is capable of quickly and thoroughly dispersing and blending materials in a range of liquid and semi-solid media.
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2 protocols using pro pk 02200d homogenizer
1
Intestinal Redox Biomarker Analysis
2
Quantification of Inflammatory Cytokines
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Before measurement, approximately 0•3 g of tissue samples (spleen, and jejunal and ileal mucosa) were homogenised in an ice-cold bath with a chilled 154 mmol/l sterile sodium chloride solution (1:9, w/v) for 30 s, using a PRO-PK-02200D homogenizer (Pro Scientific, Inc.). After that, the homogenisation was centrifuged at 4450 g for 15 min at 4°C, and the resulting supernatant was collected in five aliquots, which were immediately stored at -20°C until the subsequent analysis. The levels of interferon-γ (IFN-γ), IL-1β (IL-1β) and TNF-α (TNF-α) in the serum, spleen and intestinal mucosa samples were measured by ELISA using chicken-specific IFN-γ (catalogue no. H025), IL-1β (catalogue no. H002) and TNF-α (catalogue no. H052) quantification kits (Nanjing Jiancheng Bioengineering Institute) based on the manufacturer's instructions. The inter-and intra-assay CV were <8 and 10 %, respectively. All results were normalised against total protein level in each sample for inter-sample comparison. The total protein content in the spleen and mucosa samples was measured by Bradford method, using crystalline bovine serum albumin (Sigma-Aldrich) as the standard protein.
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