Cytokines from medium conditioned either by macrophages or MCF10A cells were analysed with human cytokine antibody array (ab133997; Abcam) according to the manufacturer's instructions. Uncultured media were tested to assess baseline signal responses. Signals were detected with chemiluminescence reaction, and densitometry analysis was performed with ImageJ software (https://imagej.nih.gov/ij/ ).
Ab133997
Manufactured by Abcam
Sourced in United Kingdom
Ab133997 is a primary antibody used for the detection of a specific target protein. The product is suitable for various immunoassay applications, such as Western blotting, immunohistochemistry, and ELISA. The antibody is manufactured by Abcam.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Quantikine kits, by R&D Systems (1 mentions)
Ab134004, by Abcam (1 mentions)
Azure imager c400, by Azure Biosystems (1 mentions)
Image lab v5, by Bio-Rad (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Amersham imager 680, by Cytiva (1 mentions)
Amersham imager, by Cytiva (1 mentions)
8 protocols using ab133997
1
Cytokine Profiling of Macrophage and MCF10A Secretome
2
Cytokine Profiling in hNEC Infection
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Cytokine profiling was performed using the human cytokine antibody array (membrane, 42 targets) (Abcam, ab133997) according to the manufacturer’s protocol using the buffers provided in the kit. Briefly, membranes were blocked with 1× blocking buffer at room temperature for 30 min and aspirated. Basal media from three biological replicates of infected hNECs were pooled in equal volumes to a final volume of 1 mL before applying to the blocked membrane. After overnight incubation at 4 °C, the membrane was washed with 20 mL wash buffer I and incubated at room temperature for 30 min with gentle shaking. Membranes were washed thrice with 1× wash buffer I and twice with 1× wash buffer II. After washing, 1× biotin-conjugated anti-cytokine antibodies were added to the membranes and incubated for 2 h at room temperature. After three washes with 1× wash buffer I and two washes with 1× wash buffer II, 1× horseradish peroxidase (HRP)-conjugated streptavidin was added and incubated for 2 h at room temperature. The membrane was washed thrice with 1× wash buffer I and twice with 1× wash buffer II and detected by buffers C and D provided. The blot was imaged using a ChemiDoc™ MP Imaging System (Bio-rad).
3
Cytokine profiling of HCoV-OC43 infection
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For 24 h, 2 × 106 MRC-5 cells were seeded in a 10 cm dish and then infected with HCoV-OC43 at MOI 0.05 for 30 h. A mock infection control was also performed. Changes in the concentrations of 42 cytokines in the culture supernatants with or without HCoV-OC43 infection were evaluated using a commercial cytokine array (Abcam, Cambridge, UK, ab133997) as per the manufacturer’s recommendations.
4
Antibody Arrays and Cytokine Quantification
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RayBio® Human Biotin Label Based Antibody Arrays - Human L-507 Array, Membrane (AAH-BLM-1A-2, RayBio®, US) was used to analyze the supernatant (conditioned media) of Huh-7 cells (control or macroH2A1 KD), according to manufacturer's instructions. A Human Cytokines antibody array membrane (Abcam, Germany) was used to analyze the supernatant (conditioned media) of HepG2 cells (control or macroH2A1 KD), according to manufacturer's instructions (ab133997, Abcam, US). Detection of IL-6 and IL-8 levels in the culture media of Huh-7 cells was performed using Quantikine® kits (Bio-Techne R&D Systems s.r.o., Prague, Czech Republic), according to manufacturer's instructions.
Nuclei protein fractions from HepG2 and Huh-7 CTL cells were isolated as previously described 23 (link), 24 (link). Primary antibodies were obtained from Active Motif (macroH2A1.1 and macroH2A1.2) and Cell Signaling Technology (H2B).
Nuclei protein fractions from HepG2 and Huh-7 CTL cells were isolated as previously described 23 (link), 24 (link). Primary antibodies were obtained from Active Motif (macroH2A1.1 and macroH2A1.2) and Cell Signaling Technology (H2B).
5
Modulation of Inflammatory Markers and ECM Components
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Conditioned medium from cancer tissue indirectly co-cultured with omentum and pre-treated with our various treatments (see material and methods above) was thawed at room temperature. Protein arrays for human growth factors and cytokines (Abcam ab133997) and components of the extracellular matrix, such as MMPs and TIMPs (Abcam ab134004), were performed as per the manufacturer’s instructions and tested for the effects of our treatments on the regulation of levels of human inflammatory markers, cytokines, and components of the extracellular matrix.
6
Cytokine Profiling using Antibody Arrays
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Human Cytokine Antibody Arrays (ab 133,996 and ab133997, Abcam) were used for the simultaneous detection of 23 resp. 42 cytokines in each CM sample according to the manufacturer’s instructions. The array membranes were incubated for 30 min at room temperature in a blocking buffer. CM samples were then incubated on the membranes overnight at 4 °C. Following one large volume wash in Wash buffer I for an extended time of 30 min, three washes in Wash buffer I and two washes in Wash buffer II, membranes were incubated in Biotin-Conjugated Anti-Cytokines overnight at 4 °C. After washing as described above, membranes were incubated in HRP-Conjugated Streptavidin overnight at 4 °C. Washed arrays were finally incubated with Chemiluminescence Detection reagents and images were captured using the Azure c400 Imager (Azure Biosystems). Pixel density (signal density) of each spot on the membrane was quantified using ImageJ—Array Analysis plugin [27 (link)].
7
Cytokine Profiling of PRAME-specific T Cells
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PRAME‐specific T cells were incubated with PRAME peptide mixture for 5 h in the presence of anti‐CD28 (L293) and CD49d (L25) antibodies. The supernatant was harvested for the detection of cytokine using dot blot cytokine antibody membrane array (Abcam ab133997, Cambridge, UK) as per manufacturer’s instructions; supernatant from end of culture T cells without restimulation was used as a control. The resultant array images were captured with Chemidoc Touch imaging system (Bio‐Rad, CA, USA) and analysed with Imagelab v5.2.1 (Bio‐Rad) and ImageJ (NIH).
8
Cytokine Antibody Array Assay
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Assay for cytokine antibody arrays was carried out as specified by the manufacturers (ab133997-Abcam).
Briefly, conditioned media prepared from MDA-MB-231 and MDA-MB-468 SCR and NLGN4X-KD cells was collected and incubated with the detection antibody cocktail. The sample/antibody mixture was then added onto the blocked membrane, containing 42 different capture antibodies. After washing, the membrane was incubated with diluted Streptavidin-HRP and Chemi Reagent Mix was added. The cytokines signals were detected by the ECL system on Amersham imager 680.
Briefly, conditioned media prepared from MDA-MB-231 and MDA-MB-468 SCR and NLGN4X-KD cells was collected and incubated with the detection antibody cocktail. The sample/antibody mixture was then added onto the blocked membrane, containing 42 different capture antibodies. After washing, the membrane was incubated with diluted Streptavidin-HRP and Chemi Reagent Mix was added. The cytokines signals were detected by the ECL system on Amersham imager 680.
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