The collected ileal tissue was mixed with the phosphate buffer at a weight (g)/volume (mL) ratio of 1:9, using the German IKA T 10 basic ULTRA-TURRAX® homogenizer at 10,000 rpm (homogenizing time of 10 s, interval of 30 s, repeated 3 times) to prepare the 10% intestinal tissue homogenate. The supernatant of the tissue homogenate was prepared by centrifuge at 4000 rpm for 10 min at 4 °C, using a high-speed refrigerated centrifuge. We used commercial kits (Shanghai Enzyme Linked Immunobiotechnology Co. Ltd.) and an automated fluorescence instrument (MultiskanM™ SkyHigh, Thermo Fisher Scientific, Waltham, MA, USA) to detect inflammatory cytokines in the ileal tissue. The detailed inflammatory cytokine indexes are the same as the serum immune indexes in Section 2.5 .
Multiskanm skyhigh
Manufactured by Thermo Fisher Scientific
Sourced in United States, China
The MultiskanM™ SkyHigh is a versatile microplate reader designed for a wide range of absorbance-based applications. It features a high-performance monochromator and a powerful xenon flash lamp to provide accurate and reliable results. The instrument can read 6- to 384-well microplates and supports multiple wavelength selection.
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6 protocols using multiskanm skyhigh
1
Intestinal Tissue Homogenate and Cytokine Analysis
2
Comprehensive Blood Profiling for Antioxidant and Immune Status
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The blood cell composition of anticoagulant blood collected in EDTA tubes was detected using a fully automated blood cell analyzer (Sysme XT-1800i, Shanghai, China). An Olympus AU640 automatic blood biochemical analyzer was used to detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The preserved serum was collected using a commercial kit (Shanghai Enzyme-linked ImmunoBiotechnology Co., Ltd., Shanghai, China) and an enzyme labeler (MultiskanM™ SkyHigh, Thermo Fisher Scientific, Waltham, MA, USA) for antioxidant and immune markers. Levels of malondialdehyde (MDA), reactive oxygen species (ROS), glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) were used as serum antioxidant indexes. Levels of tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-10 (IL-10) were taken as serum immune indicators.
3
Comprehensive Blood Biomarker Profiling
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The blood cell components in the anticoagulant blood collected from the EDTA tube were detected using an automatic hematology analyzer (SysmeXT-1800i, Shanghai, China). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using the Japanese Olympus AU640 automatic blood biochemical analyzer. We used commercial kits (Shanghai Enzyme Linked Immunobiotechnology Co. Ltd., Shanghai, China) and an automated fluorescence instrument (MultiskanM™ SkyHigh, Thermo Fisher Scientific, Waltham, MA, USA) to detect antioxidant and immune indicators in the preserved serum. Serum markers of oxidative stress included malondialdehyde (MDA), and reactive oxygen species (ROS). Serum antioxidant indicators included glutathione peroxidase (GSH-Px), catalase (CAT), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC). Serum immune indicators included tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-4 (IL-4), interleukin-6 (IL-6), and interleukin-10 (IL-10).
4
Hematological and Biochemical Analyses
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The red blood cell count (RBC), hemoglobin concentration (HGB), white blood cell count (WBC), and lymphocytes (Lym) were measured with an auto hematology analyzer (XT-1800i, Sysmex Corporation, Tokyo, Japan). The alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an automatic blood biochemical analyzer (AU640, Olympus Corporation, Tokyo, Japan). The total protein (TP) and albumin levels of serum were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) using a colorimetric method. The globulin content was obtained by subtracting the albumin value from that of the TP [25 (link)]. Total antioxidant capacity (T-AOC), activity of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and malonaldehyde (MDA) concentrations in the serum were determined by the commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) with an automated fluorescence instrument (MultiskanM™ SkyHigh, Thermo Fisher Scientific, Waltham, MA, USA).
5
Serum Biomarker Quantification Protocol
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The serum albumin, total protein and LPS levels were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China. The catalog numbers were A028-1-1, A045-3-2 and H255, respectively) by colorimetric method (UV-2550, Shimadzu, Japan). Because the total protein in serum mainly consists of globulin and albumin, the globulin content was obtained by subtracting the albumin value from that of the total protein [21 (link)]. Serum total antioxidant capacity (T-AOC), activity of total superoxide dismutase (T-SOD) and malonaldehyde (MDA) concentration were determined by the commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China. The catalog numbers were A015-2-1, A001-1-2 and A003-2-2, respectively) with an automated fluorescence instrument (MultiskanM™ SkyHigh, Thermo Fisher Scientific, Waltham, MA, USA).
6
Oxidative Stress Biomarkers Evaluation
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Samples of serum were analyzed for malonaldehyde (MDA), antioxidant biomarkers including glutathione (GSH), activities of superoxide dismutase (SOD),glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and Catalase (CAT) were determined by the commercial assay kits (Nanjing Jiancheng Bioengineering Institute, China) with an automated fluorescence instrument (MultiskanM™ SkyHigh, Thermo Fisher Scientific, Waltham, MA, United States).
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