The cells were cultured according to the scheme described in section “Cell culture” and treated for 24 hours after confluency reached about 80%. After treatment, the cells were manually detached, collected, and centrifuged at 1200 rpm for ten minutes. The protein extract was prepared with a PierceTM RIPA buffer (Thermo Scientific, USA) with protease inhibitors (Sigma-Aldrich, USA) and sonicated for three minutes with a pulse of 30 seconds on/off and an amplitude of 20%. The samples were centrifuged at 12,000 rpm for 25 minutes at 4°C. The supernatant was collected, and the total protein concentration was determined using a bicinchoninic acid kit (Sigma-Aldrich, USA). All samples from each experimental group were pooled before further procedure. Human inflammation factors were analyzed using an antibody array (ab134003; Abcam, UK). The assay was performed in accordance with the manufacturer’s instructions. The membranes were visualized using Azure Biosystem C400 (Azure, USA), and analyzed using the ImageJ software. Obtained results were normalized to the control dots.
Ab134003
Manufactured by Abcam
Sourced in United Kingdom
Ab134003 is a laboratory equipment product offered by Abcam. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
4 protocols using ab134003
1
Profiling Human Inflammation Factors
2
Cytokine Expression Profiling using Antibody Array
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Analysis of cytokine expression was performed using an antibody array for human inflammation factors (ab134003; Abcam, UK). The assay was performed in accordance with the manufacturer’s instructions. The membranes were visualised using a visible fluorescent Western blot imaging system (Azure Biosystems C400, Azure, USA) and analyzed using ImageJ software. The obtained results were normalised to the control dots. The assay was performed in accordance with the manufacturer’s instructions using lysates containing 200 μg/mL of total protein per membrane.
3
Fisetin's Anti-inflammatory Mechanism
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To validate the anti-inflammatory role of fisetin, modulated expression of different anti-inflammatory proteins after fisetin treatment at 50 µM was compared with the untreated control. Human inflammation antibody array was carried out, which was procured from Abcam (ab134003; Cambridge, UK). To perform the array, firstly, protein quantitation of the cell lysate was performed by BCA assay. After blocking the membrane by blocking buffer for 30 min, each membrane was incubated with ~250 µg of lysate overnight on a rocking platform at 4 °C, followed by washing with wash buffer I and II to discard any unbound proteins. After the washes, the nitrocellulose membranes were labelled with biotinylated antibody. Finally, HRP-Streptavidin was added and left on the rocking surface for 2 h followed by washing and the subsequent addition of 500 µL detection buffer mixture C and D. Within 5 min, the membrane was exposed to chemiluminescent detector gel doc system (Bio-Rad Laboratories, Richmond, California, USA). Image Lab software, version 6.0.1, Bio-Rad was used to analyse the data.
4
Cytokine Expression in HCV Macrophages
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