Cell culture supernatant was collected from ISG20-overexpressing and control of SK-Hep1 for 24 h. The angiogenic factors secreted in supernatant was measured using angiogenesis array kit (R&D systems, Minneapolis, MN), according to the manufacturer's instructions. After thorough washing, blots were further incubated with HRP-conjugated secondary antibody dissolved in blocking solution. Signals were detected via Chemi Reagent Mix and recorded on X-ray film.
Angiogenesis array kit
Manufactured by R&D Systems
Sourced in United Kingdom
The Angiogenesis Array Kit is a tool for the simultaneous detection and quantification of multiple angiogenesis-related proteins in a single experiment. It provides a comprehensive analysis of angiogenic factors in biological samples.
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Lab products found in correlation
5 protocols using angiogenesis array kit
1
Angiogenic Factors Secretion Profiling
2
Angiogenesis and Cytokine Profiling of ACS Cells
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Cells from ACS were maintained in culture for up to 7 days with daily medium-change of the Epilife™ medium either supplemented (‘complete’) or non-supplemented with HKGS (see Section 2.1 ). On days when the ACS culture medium was to be collected, cells were always incubated in non-supplemented Epilife™ medium for 24 h prior to collection, and this was denoted as ACS conditioned medium (CM). Detection of analytes released in vitro from collected conditioned medium (at the indicated time points following culture initiation) from ACS-derived cultures was performed using an angiogenesis array kit (#ARY007) and a cytokine array kit (#ARY005B) (both from R&D Systems, supplied by Bio-Techne, Abingdon, United Kingdom). Array processing was carried out following the manufacturer’s instructions, with the exception of the supplied streptavidin secondary antibody being replaced by IRDye® 800CW Streptavidin antibody (#926–32230, LI-COR Biosciences, Cambs, United Kingdom), to enable fluorescence-based detection on an Odyssey™ Infra-red Imaging system (LI-COR), as detailed (Ibraheem et al., 2018 (link)).
3
Detecting Soluble Proteins in MSCs
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To detect soluble protein, MSCs Pml+/+ or Pml−/− were plated at the same numbers, and the supernatant was collected after a few days of culture in hypoxic conditions. Collected supernatants were analyzed within Mouse Cytokine array panel A array kit and Angiogenesis array kit (R&D Systems), following manufacturer instructions.
4
Multiplex Cytokine and Angiogenesis Analysis
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The Human Cytokine Array Kit and Angiogenesis Array Kit (R&D Systems) evaluated 36 cytokines, chemokines, acute phase proteins, and 55 angiogenesis-related proteins. The experiments were carried out according to the manufacturer's protocol. For screening, plasma samples were mixed with detection antibodies and incubated with arrays containing duplicate spots of capture-labelled antibodies. After washing, the arrays were incubated with streptavidin-conjugated horseradish peroxidase. The chemiluminescent substrate was added, and signals were detected by a MicroChemi analyzer (DNR Bioimaging System). Densitometric studies of the averaged pixel density of duplicate spots were carried out using Quantity One software (Bio-Rad). Relative cytokine levels were calculated compared to control time point 1. An average background signal from negative controls was subtracted from each spot during the analysis. Positive control spots were also included on each membrane to ensure the repetitiveness of each assay.
5
Quantifying Angiogenic Secretome of LO-EPC
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The levels of multiple cytokines/chemokines and angiogenic factors in plasma were measured using a commercial Cytokine Antibody Array kit (R&D Systems, Abingdon, UK) or Angiogenesis Array kit (R&D Systems) according to the manufacturer's instructions. The array data were analyzed by Image J software (freeware, http://rsbweb.nih.gov/ij/ ) and expressed as pixel density. To evaluate the secretion of angiogenic factors in LO-EPC in vitro, cells were cultured in growth-factor-free basal medium EBM with 10% FBS for 24 h. The supernatant was then collected, and the levels of multiple angiogenic factors measured.
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