Circulating levels of tH3, H3K27ac, and H3K4me3 were measured using enzyme-linked immunosorbent assay (ELISA) kits (Epigentek; Farmingdale, NY) and read on BioTek Synergy H1 (BioTek Instrument Inc., Winooski, VT) at 450 nm with 570 nm correction. CRP was also measured with an ELISA kit (Sino Biological; Wayne, PA) and read on BioTek Synergy H1 (BioTek Instrument Inc., Winooski, VT) as specified above. TNFα, IL-6, and HSP-60 were measured using a multiplex assay (Thermo Fisher, Philadelphia, PA) and analyzed on 3D FlaxAmp (Thermo Fisher, Philadelphia, PA).
Multiplex assay
Manufactured by Thermo Fisher Scientific
Sourced in Israel, United Kingdom, United States
The Multiplex assay is a laboratory technology that enables the simultaneous measurement of multiple analytes or targets in a single sample. It is designed to provide efficient and high-throughput analysis of diverse biomolecules, such as proteins, nucleic acids, or metabolites. The core function of the Multiplex assay is to facilitate the simultaneous detection and quantification of multiple analytes in a cost-effective and time-saving manner.
Automatically generated - may contain errors
4 protocols using multiplex assay
1
Epigenetic and Inflammatory Biomarkers
2
Murine Model of Allergic Asthma
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BALB/c female mice were sensitized with 100 µg OVA in 2 mg aluminum hydroxide by intraperitoneal injections (200 µL) performed once a week (on days 0, 7 and 14), followed by OVA challenge (exposure for 20 min to nebulized 4% OVA) for four consecutive days (days 25, 26, 27 and 28). EXO-mCD24 was administered in an inhalation cage for 20 min on days 22, 23, and 24, and/or during the challenges on days 26, 27, and 28. Blood was collected 24 h after challenge (day 29). Mice were sacrificed, and BALF and lung tissue were obtained. Differential cell counts (% of BALF cells) was analyzed by flow cytometry; the multiplex assay was used for the evaluation of cytokines, chemokines, and IgE (ThermoFisher, Rhenium, Modiin, Israel).
The Mann–Whitney U statistical test was used to compare between the animal groups.
The Mann–Whitney U statistical test was used to compare between the animal groups.
3
Quantitative Analysis of MMPs and Cytokines
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Concentrations of MMP-2 and MMP-9 in plasma were quantitated with a commercial enzyme-linked immunosorbent assays (ELISA: R&D® Systems, Minneapolis, USA). Levels of IL-4 and TGF-β1 were quantitated with a Multiplex® Assay (Invitrogen, Paisley, UK) using the Luminex® 100™ instrument. All reaction steps were performed according to the manufacturers’ protocols.
4
Secretome Characterization of Cell Types
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For the characterization of the secretomes of both cell types and coculture, 30 growth factors, cytokines, and chemokines were quantified using either ELISA or multiplex assay (Invitrogen, Grand Island, NY, USA) following the manufacturer’s instructions. A Luminex Labscan 100-plate reader (Luminex Corporation, Austin, Texas, USA) was used for determination. The molecules quantified by multiplex were the following: MIP1 alpha, IL-2, IL-6, TIMP-1, IL-8, IL-10, IL-12 P70, IL-1 RA, RANTES, GM-CSF, leptin, HGF, MMP-3, MCP1, BNGF, EGF, adiponectin, TNF alpha, MMP-1, TRAIL, FGF-2, PDGF-BB, and VEGF-A. For quantification of IGF-1, BMP-6, IL-1β, IL-4, TGF-β1, TGF-β3, and VEGF-C, commercial ELISA kits were employed (DuoSet ELISA kits, R&D, Minneapolis, MN, USA), and the manufacturer’s instructions were followed. Readings were performed on an iMark plate reader (BioRad, Hercules, CA, USA), and absorbances were measured at 450 and 570 nM.
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