Human adipokine array kit (Cat. #ARY024, R&D systems) was used to detect up to 58 human adipokines in serum from breast cancer patients and normal subjects. Antibody-loaded membranes were incubated with 80µl serum and analyzed according to the manufacturer’s instruction at the same condition. The images were captured and acquired using Azure C300 Ultimate Western Blot Imaging System. Data was transferred by Image J software, analyzed by subtracting the average background signal from each spot and finally compared to the average of reference spots on different arrays to determine the relative levels of interested proteins.
Human adipokine array kit
Manufactured by R&D Systems
The Human Adipokine Array Kit is a multiplex assay designed to detect the relative levels of 40 different adipokines in human cell culture supernatants, plasma, or serum samples. The kit utilizes a membrane-based antibody array format to provide a semi-quantitative measurement of multiple analytes simultaneously.
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Lab products found in correlation
Human angiogenesis array kit, by R&D Systems (1 mentions)
Proteome profiler antibody array, by R&D Systems (1 mentions)
Human soluble receptor array kit, by R&D Systems (1 mentions)
Ez capture mg, by ATTO Corporation (1 mentions)
Amersham imager 600 system, by GE Healthcare (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
6 protocols using human adipokine array kit
1
Profiling Adipokines in Breast Cancer
2
Serum Proteomic Profiling of Angiogenesis, Receptors, and Adipokines
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Proteomic analyses of serum proteins were carried out using Proteome Profiler antibody arrays (R&D Systems, Minneapolis, MN), namely the Human Angiogenesis Array kit (ARY007), Human Soluble Receptor Array kit (ARY012), and Human Adipokine Array kit (ARY024). Each examination was performed according to the respective protocol. Briefly, serum samples for use with each kit were applied to nitrocellulose membranes with bound capture antibodies for target proteins and incubated for 24 hours in a refrigerated room followed by removing serum from the membrane and assaying immediately. Samples were analyzed using a LAS system (Ez‐Capture MG; ATTO Corp., Tokyo, Japan). Measured signals are presented as percentage of the negative control (0%) and positive control (100%). We selected proteins in an unbiased manner that met the following criteria: (1) significant difference (P < 0.05) in rate of change in value between the R group and NR group; (2) exhibit >1.5‐fold difference between before and 1 month after SFN treatment initiation; and (3) measured value >5%.
3
Adipokine Profiling of MSCs
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MSCs were cultured in 12‐well plates at a density of 2 × 104 cells/cm2. After 3 days of ROA knockdown, culture supernatants were collected from each group and analyzed using a Human Adipokine Array Kit (R&D Systems) as instructed. Each assay was performed with 500 μL of culture supernatant. The pixel density of each spot was measured by ImageJ (NIH). Mean pixel densities were normalized to the reference spots.
4
Adipose-Conditioned Medium Impacts Myogenesis
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To generate adipose-conditioned medium (ACM), SAT samples were incubated in differentiation medium at a ratio of 1 g of tissue to 10 mL of medium for 24 h. Larger samples were divided into segments of ~1 g to standardize the surface area of adipose tissue. The ACM was sterile filtered and stored at −80 °C. Prior to use, ACM from numerous patients (normal adiposity: n = 18; excess adiposity: n = 15) was combined to generate a stock of normal adiposity and a stock of excess adiposity ACM, which were matched for mean age and sex distribution (Table 2 ). The relative abundance of 58 adipokines within both pooled ACM stocks was assessed using a commercially available human adipokine array kit (R&D Systems, #ARY204).
Upon reaching > 90% confluence, myogenic cultures from n = 4 healthy participants (BMI range: 19.8–24.5 kg·m−2) were switched to either unconditioned, normal adiposity ACM or excess adiposity ACM for differentiation. Freshly-thawed ACM was diluted 1:2 with fresh differentiation medium and was renewed every 48 h for 6 days [36 (link)].
Upon reaching > 90% confluence, myogenic cultures from n = 4 healthy participants (BMI range: 19.8–24.5 kg·m−2) were switched to either unconditioned, normal adiposity ACM or excess adiposity ACM for differentiation. Freshly-thawed ACM was diluted 1:2 with fresh differentiation medium and was renewed every 48 h for 6 days [36 (link)].
5
Adipokine Profiling from Cell Supernatants
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The Human Adipokine Array Kit (Cat#ARY024, R&D Systems) was used to measure the levels of adipokines in supernatants in accordance with the manufacturer's instructions. Nitrocellulose membranes, spotted with capture and control antibodies, were incubated in array block buffer at room temperature for 1 h. Samples were mixed with reconstituted human adipokine detection antibody cocktail and incubated at room temperature for 1 h. Next, the mixtures were incubated with the membrane overnight at 4 °C. After washing 3 times, the membrane was incubated in 2 ml diluted streptavidin-HRP for 30 min at room temperature. At last, the membrane was exposed by the Amersham Imager 600 system (GE Healthcare, USA).
6
Adipokine Profiling of Tissue Lysates
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The Human Adipokine Array Kit (ARY024 from R&D Systems) was used according to the manufacturer’s instructions. Tissue lysates were prepared. Briefly, the membranes containing adipokine antibodies were blocked and then incubated with tissue lysates (50 µg) for 2 h at room temperature, washed in Wash Buffer, and then incubated with biotin‐conjugated antibodies for 2 h and with a horseradish peroxidase‐linked secondary antibody for another 2 h. The membranes were incubated with chemiluminescent substrate. The chemiluminescence was detected by ChemiDoc XRS System (Bio‐Rad, Hercules, CA, USA).
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