Cytokine profiling of the cells was performed using the Human Inflammation Antibody Array- Membrane (Abcam product#ab134003). This array analyses human protein samples for 40 different inflammatory cytokines. Array membranes, were incubated for 30 min in 2 ml of blocking buffer supplied with the kit. Following this, they were further incubated for 60 min at room temperature with 200µg of protein from each cell sample. One ml of Biotin-conjugated Anti-Cytokines were then added to each membrane after a thorough wash. This was succeeded by an overnight incubation at 4°C and then incubated with a 1:2000 dilution of HRP-Conjugated Streptavidin for 60 min at room temperature. The proteins were detected by chemiluminescence western blot and signals were captured on x-ray films (Denville Scientific, MA, USA), scanned at high resolution, and quantified using ImageJ software.
Human inflammation antibody array membrane
Manufactured by Abcam
Sourced in United States, United Kingdom, France
The Human Inflammation Antibody Array - Membrane is a lab equipment product that detects the simultaneous expression of multiple inflammation-related proteins in human samples. It provides a comprehensive, semi-quantitative analysis of inflammatory markers.
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4 protocols using human inflammation antibody array membrane
1
Cytokine Profile Analysis Using Antibody Array
2
Multiplex Inflammation Profiling in Resistant Cells
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For the simultaneous detection of 40 Human inflammatory factor concentrations in melatonin-treated HepG2/dox-resistant cell lysates, human inflammation antibody array- membrane (cat# ab 134,003, Abcam, USA) was used (Lu et al. 2020 (link)). The manufacturer protocol was followed. Briefly, after blocking the membrane array with 1X blocking buffer, the membrane was incubated with the sample lysate (140 µg) overnight at 4 °C. The membrane array was washed thoroughly with wash buffers I, and II that were supplied in the kit. The membrane was then incubated with 1 × Biotin-conjugated anti-cytokines overnight at 4 °C. After washing steps, the membrane was incubated with 1 × HRP (horseradish peroxidase)-conjugated streptavidin for 2 h at RT. The Chemiluminescence signals were detected by a CCD (charged-coupled device) camera of a chemiluminescence imager (UVP, UK) to digitally visualize protein spots on the developed membranes. Spot densitometric analysis of the arrays was performed using Visionworks ls (Analytik Jena, Germany). Then, the background signals were subtracted, and normalization to the positive control was calculated before comparing analyte-by-analyte to determine relative differences in cytokine expression in each sample.
3
Cytokine Profiling of Nanoparticle-Treated HFFF2 Cells
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In order to determine the cytokine expression in HFFF2 cells, a Human Inflammation Antibody Array – Membrane (ab134003, Abcam, Cambridge, UK) for 40 targets was performed. The HFFF2 cells were treated with nanoparticles and farnesol complexes (at concentrations of 10 µg/mL for nanoparticles and 1% for farnesol) and incubated for 24 h. Then, the cells were trypsinized and harvested, and the protein extract was prepared using a TissueLyser LT (Qiagen, Hilden, Germany) at 50 Hz for 10 min with frozen metal balls. After that, the samples were centrifuged at 13,000× g for 10 min at 4 °C, and the pellets were collected. The protein concentration was measured using the BCA Protein Assay (Thermo Scientific) according to the manufacturer’s protocol. The cytokine array was conducted in accordance with the manufacturer’s instructions, and the membranes were imaged using Azure Biosystem C200 (Dublin, CA, USA). The images were then analyzed with ImageJ software (version 1.50e, National Institutes of Health, USA) using the Protein-Array Analyzer plugin.
4
Cytokine Profiling in EC-Porphyromonas gingivalis
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To evaluate the impact of M101 treatment on the protein expression of cytokines in EC infected by P. gingivalis (MOI = 100), a Human Inflammation Antibody Array—Membrane (Abcam ab134003, Paris, France) was used according to manufacturer’s instructions. Prior to the experiments, 2 × 105 EC previously infected with P. gingivalis MOI = 100 were treated with M101 at 1 g/L for 6 h. Then, supernatants were collected and subjected to proteomics analysis (see Supplemental File).
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