Conditioned medium from confluent BT-549 and Hs578t cells stably expressing either IRX2 or empty vector were tested for chemokine secretion by chemokine antibody array following the manufacturer’s instructions (R&D Systems Proteome Profiler™ Human Chemokine Array Kit). Estimation of normalized signal intensity was done using ImageJ software.
Proteome profiler human chemokine array kit
Manufactured by R&D Systems
Sourced in United States, United Kingdom
The Proteome Profiler Human Chemokine Array Kit is a multiplex assay designed to detect the relative levels of 31 different human chemokines in a single sample. The kit uses a membrane-based antibody array format to enable simultaneous detection of multiple analytes from a single sample.
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Lab products found in correlation
Nf κb phospho antibody array, by Full Moon BioSystems (1 mentions)
Minisart 0.2 μm syringe filter, by Sartorius (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Amersham hyperfilm ecl, by GE Healthcare (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Human phospho kinase array kit, by R&D Systems (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Secondary goat anti mouse apc, by BioLegend (1 mentions)
Cellquest pro software, by BD (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Chemi reagent mix, by R&D Systems (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Pierce coomassie bradford protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Proteome profiler human xl cytokine array kit, by R&D Systems (1 mentions)
Proteome profiler human cytokine array panel a, by R&D Systems (1 mentions)
Super rx n film, by Fujifilm (1 mentions)
15 protocols using proteome profiler human chemokine array kit
1
Chemokine Secretion Profiling of IRX2-Expressing Cells
2
Profiling Ror2-Silenced MSC Secretome
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Conditioned media obtained from Ror2‐silenced MSCs were analyzed using the Proteome Profiler Human Chemokine Array Kit (ARY017; R&D Systems), according to the manufacturer's instructions. Briefly, array membranes, spotted with capture antibodies to specific target proteins, were incubated overnight at 4°C with the conditioned media pretreated with a cocktail of biotinylated detection antibodies. Then, membranes were washed and incubated with streptavidin–HRP for 30 min at room temperature. After washing the membranes, captured proteins on the membranes were visualized using chemiluminescent detection reagents. Supplementary materials and methods are described in Data S1.
3
Chemokine and NF-κB Pathway Analysis in Cancer Cells
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For the chemokine array, we cultured 1 × 106 cancer cells in 1 mL of D0 medium for 16 hours at 37°C. Then we collected the supernatants and cell lysates so that we could detect the chemokines using a Proteome Profiler Human Chemokine Array Kit (R&D Systems, ARY017).
For the NF-κB Phospho Antibody Array, we suspended 2 × 106 cancer cells in 2 mL of D0 medium and seeded the cells on collagen I-coated 6-well culture plates for 3 hours. The cell lysates were then collected, and we detected the NF-κB pathway using an NF-κB Phospho Antibody Array (Full Moon BioSystems, KAS02).
For the NF-κB Phospho Antibody Array, we suspended 2 × 106 cancer cells in 2 mL of D0 medium and seeded the cells on collagen I-coated 6-well culture plates for 3 hours. The cell lysates were then collected, and we detected the NF-κB pathway using an NF-κB Phospho Antibody Array (Full Moon BioSystems, KAS02).
4
Profiling Melanoma Chemokine Secretome
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Melanoma cell lines were grown to 100% confluency in RF10 prior to replacing the media and culturing cells for a further 3 days. The conditioned media was collected, centrifuged to exclude any cells, filtered with a Minisart 0.2 μm syringe filter (Sartorius AG, Gottingen, Germany) and stored at −80°C prior to 1 ml being added to the Proteome Profiler Human Chemokine Array Kit (R&D Systems, Minneapolis, MN, USA). The membrane was visualized using the LAS-4000 (FujiFilm, Tokyo, Japan). Quantification of the dots on the membrane was performed using ImageQuant™ TL (GE Healthcare Life Sciences, Marlborough, MA, USA).
5
Comparative Proteome and Kinase Profiling
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Proteome Profiler Human Chemokine Array Kit (R&D Systems) was used to compare 31 different chemokines in the CM of KYSE410-CON, KYSE410-SRGN, and KYSE410-∆GAG cells according to the manufacturer's instructions. Human Phospho-Kinase Array Kit (R&D Systems) was used to compare 45 different kinases in cell lysates of KYSE30-CON and KYSE30-MDK. Immunoreactive signals were detected using Clarity™ Western ECL Substrate (Bio-Rad) and exposed to Amersham Hyperfilm ECL (GE Healthcare, Chicago, IL, USA).
6
Chemokine Profiling of Conditioned Media
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The conditioned media from HCC cells cultured in high glucose and normal glucose conditions (HG-CM and NG-CM) for 48 h were collected respectively, and their differentially expressed chemokines were analyzed using a Proteome Profiler™ Human Chemokine Array Kit (R&D Systems, Cat. No. ARY017) in accordance with the instructions and the published studies [[72] (link), [73] (link), [74] (link)]. Briefly, the conditioned media was added with reconstituted detection antibody cocktail and incubated at room temperature for 1 h. Subsequently, the sample/antibody mixtures were administered to each array and incubated at 4 °C overnight. After washing, the array was further incubated with diluted Streptavidin-HRP for 30 min, and then reacted with Chemi Reagent Mix. Signals of the array were captured using the Bio-Rad Chemi Doc XRS imaging system (Hercules), and their signal intensity was quantified using ImageJ software. The relative expression of chemokine was normalized to the average intensity of the positive control spots on the same array. The differentially expressed chemokines between HG-CM and NG-CM were defined using a fold change cut-off of >1.2 or <0.8.
7
Profiling Chemokine Secretion in Cell Culture
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The cell culture media was harvested after 48 h and processed for profiling chemokines using the Proteome Profiler Human Chemokine Array Kit (R&D system, Minneapolis, MN), which detects a panel of 31 chemokines (Table 2 ). The array membranes were reacted with the mixture of conditioned media and the antibody cocktail for 18 h at 4 °C. After 3 washes, they were briefly incubated with secondary antibodies conjugated with horseradish peroxidase (HRP). The membranes were then exposed to HRP substrate for 30 min. The intensity of the reaction was quantified using a BIO-RAD ChemiDoc MP Imaging System (Bio-Rad, UK); the pixel densities on developed X-ray film were analysed using Image Lab software (Bio-Rad, UK). The supernatants were assayed for MCP-1 concentrations with a human MCP-1 ELISA kit (Biolegend, UK).
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Chemokines detected with the Proteome Profiler Human Chemokine Array
8
Profiling Human Chemokine Secretion
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Proteome Profiler™ Human Chemokine Array Kit (R&D Systems, Abingdon, UK, Cat. No. ARY017) was used. Samples were pooled supernatants from ESC cultures and the manufacturer’s protocol was followed without deviation. The data are expressed as percentage of positive control of each membrane and pixel density was measured using ImageJ.
9
Analyzing Host Cell Response to Viral Infection
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For protein microarray analysis, 2 · 105 HUVEC and HSaVEC were infected with RV at an MOI of ten in two independent experiments. Supernatants were collected 48 hpi and analyzed for the presence of chemokines using the Proteome Profiler Human Chemokine Array Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. For flow cytometry analysis, 1°105 HUVEC and HSaVEC were infected with RV at an MOI of 5 and detached with trypsin at various time points post infection. Following fixation with 2 % paraformaldehyde for 20 min, cells were permeabilized with 0.5 % saponine and stained with a RV anti-capsid antibody (Meridian Life Science, USA) and a secondary goat anti-mouse APC (Biolegend, USA). Flow cytometry data were acquired on a FACSCalibur flow cytometer (BD Biosciences, USA) using Cell Quest Pro software (BD Biosciences).
10
Protein Expression Profiling of Melanoma Cell Co-Culture
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Protein expression analyses of HHSEC cells were performed before and after co-culturing with different melanoma cell lines (WM983A, WM793B, WM1366, WM1361, and WM278), as described in detail before [66 (link)]. RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 20 µL protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used for extraction, and the protein concentration was determined using the Pierce™ Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). The Proteome Profiler Human Chemokine Array Kit was used to determine the expression of 31 different chemokines, and the Proteome Profiler Human XL Cytokine Array Kit was used to analyze 105 cytokines simultaneously (R&D Systems, Inc., Minneapolis, MN, USA). All the necessary reagents and the array procedure were performed according to the manufacturer’s detailed protocol. The labeled proteins were detected and visualized by the Azure c300 Chemiluminescent Imaging System (Dublin, CA, USA) using Chemi Reagent Mix (R&D Systems Inc., Minneapolis, MN, USA). Data were analyzed with AzureSpot (version: 2.2.167) software. The intensity of the positive control (reference spot) was considered 100%.
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