After 5 days of culture, media were changed for the last 24 hours of culture to be collected and analyzed for the presence of cytokines. All CDC cultures were in equivalent cell number/volume of media ratio. Media were centrifuged at 2000 rcf for 5 minutes and then stored at −80°C until analysis. Media were analyzed by a membrane-based ELISA (RayBio® Human Cytokine Antibody Array 5; RayBiotech, Norcross, GA, USA), according to the manufacturer's instructions. Densitometric analysis was performed by ImageJ software, and data is presented as optical density values normalized to the assay's internal positive control.
Raybio human cytokine antibody array 5
Manufactured by RayBiotech
Sourced in United States
The RayBio Human Cytokine Antibody Array 5 is a multiplex assay that allows for the simultaneous detection and quantification of 80 different human cytokines, chemokines, and growth factors in a single experiment. The array utilizes the sandwich immunoassay principle to capture and detect target analytes from biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Raybio human chemokine antibody array 1, by RayBiotech (1 mentions)
Ibright imaging system, by Thermo Fisher Scientific (1 mentions)
Cxcl5, by R&D Systems (1 mentions)
Recombinant human oncostatin m, by R&D Systems (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Bio plex cell lysis kit, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
9 protocols using raybio human cytokine antibody array 5
1
Cytokine Profiling in Cell Culture
2
Cytokine Detection in Conditioned Media
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The presence of soluble factors in the CM were detected using the RayBio Human Chemokine Antibody Array 1 (Cat# AAH-CHE-1) for tumoral CM and RayBio Human Cytokine Antibody Array 5 (Cat# AAH-CYT-5) for MSC CM (Ray Biotech, Inc.) according to the manufacturer's protocol. The intensities of signals were quantified by densitometry with ImageJ software (National Institute of Health, USA) and positive controls were used to normalize the results from different membranes.
3
Cytokine Profiling of VP-Treated MDA-MB-231 Cells
4
Cytokine Profiling of VP-Treated MDA-MB-231 Cells
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MDA-MB-231 cells were grown at low density in culture and treated with 1 μM VP for 24 h in serum-free DMEM. Conditioned media was collected and concentrated as described earlier. A RayBio® Human Cytokine Antibody Array 5 (Raybiotech, Norcross, GA) was used according to the manufacture's protocol.
5
Cytokine Secretion Profiling of PSs and PDCs
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After 5 days of culture on poly‐d ‐lysine in PGM and after thorough PBS washing, serum‐free CEM medium was conditioned for 24 hours by PSs and thus contained only proteins secreted by the cells. Serum‐free CEM was also conditioned by PDCs in an equivalent ratio of cell number to media volume. Medium was centrifuged at 2,000 rcf for 5 minutes and then stored at −80°C until analysis. Media were analyzed by membrane‐based enzyme‐linked immunosorbent assay (RayBio Human Cytokine Antibody Array 5; Ray Biotech, Norcross GA, http://www.raybiotech.com ), according to the manufacturer’s instructions. Densitometric analysis was performed by ImageJ software, and data were presented either as optical density values or PS‐to‐PDC ratio.
6
Cytokine Profiling of p53 and Nox4 Variants
7
Cytokine Analysis of Keloid Fibroblasts
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The cells were seeded in a 100-mm plate and treated with 10 nM dasatinib (conditioned medium (CM)) or vehicle for 48 h. The cells were reseeded in DMEM without growth factors for 96 h and incubated at 37°C. CM was used for downstream analysis of keloid fibroblasts. The remaining CM was harvested and stored at −80°C for protein cytokine array analysis using a RayBio Human Cytokine Antibody Array 5 (RayBiotech, Peachtree Corners, GA, USA). Membranes were treated and analysed according to the manufacturer’s protocol. Densitometric analyses were performed using ImageJ software. Keloid fibroblasts were treated with 10 ng/mL CXCL5, CCL5, CCL7, and CCL8 (R&D Systems, Minneapolis, MN, USA) for 24 h.
8
Macrophage Polarization Markers Assay
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Anti-VHL polyclonal antibody and monoclonal antibodies against VEGF, CD206, CD163 and β-Actin were purchased from Santacruz (USA). Recombinant human OncostatinM, recombinant human Eotaxin, anti-Oncostatin M neutralizing antibody and anti-Eotaxin Neutralizing antibodies were procured from R&D Systems (USA). RayBio human cytokine antibody array (5) was procured from Ray Biotech (USA). FITC conjugate of anti-CD206 antibody was procured from BD Biosciences. Alexafluor 488 and Alexafluor 555 conjugates of anti-mouse and anti-rabbit IgG were procured from invitrogen (USA). HRP conjugates of rabbit and mouse IgG were purchased from Cell Signaling Technology (USA) 8μm polycarbonate (PCF) cell culture inserts were purchased from Millipore (USA). Human plasma fibronectin and Phorbol 12-myristate 13-acetate (PMA) were procured from GIBCO-Invitrogen Corporation (USA) and Calbiochem (USA) Respectively.
9
Quantifying Cytokine Profiles in EPCs
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EPCs were lysed using a Bio-Plex Cell Lysis Kit (Bio-Rad, Hercules, CA, USA). Qualitative assessment of 80 cytokines in cell lysates was performed with RayBio Human Cytokine Antibody Array 5 (RayBiotech, Norcross, GA, USA). Cytokine arrays were analyzed by densitometry using Bio-Rad Quantity One software. Expression levels were displayed as a heat map.
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