Homogenates were probed for 41 unique analytes using a commercially available, multiplex assay per the manufacturer’s protocol (Catalog# HCYTMAG-60K-PX41, Millipore Sigma, Burlington, MA, USA) and as previously described (16 (link)). The multiplex assay was selected for its large inflammatory panel to provide data for exploratory analysis since the particular proteins that would contribute to a multivariate model were unknown, and the promiscuity of a customized bead could be avoided. Data were acquired with the MAGPIX system (Luminex Corporation, Austin, TX, USA) and analyzed using MILLIPLEX Analyst 5.1 (Millipore Sigma, Burlington, MA, USA). Individual protein concentrations were normalized to total protein concentration to yield individual analyte concentrations in pg/mg of total protein.
Hcytmag 60k px41
Manufactured by Merck Group
Sourced in United States, Germany
HCYTMAG-60K-PX41 is a laboratory equipment product manufactured by Merck Group. It is a magnetic bead-based separation system designed for high-throughput cell isolation and purification applications. The core function of this product is to facilitate the separation and enrichment of target cells from complex biological samples using magnetic beads coated with specific antibodies or ligands.
Automatically generated - may contain errors
Lab products found in correlation
Milliplex analyst 5, by Merck Group (3 mentions)
Hcyp2mag 62k, by Merck Group (2 mentions)
Milliplex map, by Merck Group (2 mentions)
Milliplex analyst, by Merck Group (2 mentions)
Tissuelyser, by Qiagen (1 mentions)
Protease phosphatase inhibitor, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Hcytomag 60k, by Merck Group (1 mentions)
Milliplex human cytokine chemokine magnetic bead premixed 41 plex kit, by Merck Group (1 mentions)
Enzyme linked immunosorbent assay kit, by Cloud-Clone (1 mentions)
Hmhmag 34k, by Merck Group (1 mentions)
Milliplex map assays, by Merck Group (1 mentions)
Magpix, by Merck Group (1 mentions)
14 protocols using hcytmag 60k px41
1
Multiplex Analysis of Inflammatory Proteins
2
Nicotine-Induced Cytokine Profiling
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Supernatants from cell culture were collected after treatment with or without nicotine for 48 h as noted above. For the multiplex protein assay, supernatants and assay were prepared per the manufacturer protocol (HCYTMAG-60K-PX41, Millipore Sigma, Burlington, MA, USA). A total of 25 µL of supernatant was used in each well. Data were acquired using the MAGPIX system (Luminex Corporation, Austin, TX, USA) and analyzed using MILLIPLEX Analyst 5.1 (Millipore Sigma, Burlington, MA, USA). For ELISA, supernatants and reagents were added to the 96-well plate per the manufacturer’s protocol (50-246-338, Fisher Scientific, Hampton, NH, USA). The optical density was read at 450 nm.
3
Multiplex Profiling of PDAC Tumors
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Tissue samples were obtained from patients with confirmed PDAC through a prospectively maintained tissue bank at the University of Florida (IRB201600873) from October 14 2011 to April 23, 2018. Patients were excluded if they did not have matched peripheral blood immunophenotyped samples. Tumor samples were stored immediately following surgical resection and flash frozen in liquid nitrogen within twenty minutes of resection and stored at −80 °C. At the time of processing, tumors underwent homogenization and were then probed for IL-1 analytes using a commercially available multiplex assay per the manufacturer's protocol (Catalog# HCYTMAG-60K-PX41, Millipore Sigma, Burlington, MA, USA) and as previously described [11] (link).
4
Inflammatory Cytokine Profiling of Surgical Tissue Samples
5
Cytokine and Protein Profiling by Luminex
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Cytokine and soluble protein levels in patient serum were measured using Luminex microbead immunoassays that cover 75 analytes in four multiplex panels (catalog numbers: HCYTMAG-60K-PX41; HCP2MAG-62K-PX23; HCYP3MAG-63K-11; HSTCMAG-28SK-17; Millipore Sigma, Billerica, MA). Luminex reagents were purchased from Millipore Sigma, and the profiling assays were carried out following manufacturer’s instructions for sample preparation, incubation times and conditions, and reader settings. The Bristol Myers Squibb in-house established protocol was followed for sample/reagent volume and 384-well assay format.
6
Multivariate cytokine profiling in BAL and plasma
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In-house assays: Cytokine levels in matched BAL fluid and plasma collected from patients were measured using the multiplexed human cytokine/chemokine magnetic bead kits from Millipore (HCYTMAG-60K-PX41 and HCYP2MAG-62K) according to manufacturer’s protocol (HCYTOMAG-60K Rev. 18-MAY-2017). Briefly, frozen BAL and plasma samples were thawed, spun at 500rcf for 5 minutes to clarify, and 25uL of sample was added to 25uL of premixed magnetic beads (minus the beads for RANTES, PDGF-AA, and PDGF-AB/BB, which were excluded from analysis) in the provided 96 well plate, incubated at 4°C for 4 hours, washed, and then sequentially labeled with 25uL of detection antibodies followed by 25uL of streptavidin-phycoerythrin prior to analysis using a Luminex® 200 system. Raw MFI, bead counts, and standard concentrations were exported and analyzed as described below. CRO assays: Remaining assays were performed by Eve Technologies (Calgary, Alberta, Canada). Samples were thawed and aliquoted at 100μL, frozen and shipped to the CRO on dry ice. The Human Cytokine/Chemokine 71-Plex Discovery Assay (HD71) was then performed on each sample. Custom outputs containing raw MFI values, standard curve concentrations, and bead counts for processing as described below.
7
Cytokine/Chemokine Profiling Assay
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Dulbecco’s phosphate-buffered saline (dPBS, cat 14190), Corning CellBIND® 384 well plates (cat # CLS3764), and Milliplex Human Cytokine/Chemokine MAGNETIC BEAD Premixed 41 Plex Kit. (Millipore HCYTMAG-60K-PX41).
8
Quantifying Cytokine and Chemokine Levels
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Cytokine and chemokine levels were measured using a Milliplex human cytokine/chemokine magnetic bead premixed 41-plex kit (catalog number HCYTMAG-60K-PX41; Millipore) on the plasma collected from the EVD cohort, as previously described (74 (link)). More details can be found in Text S1 .
9
Multiplex analysis of adipokines and cytokines
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The concentration of leptin was determined on the multiplex analyzer MAGPIX (Luminex, Austin, TX, USA) using xMAP® Technology with the use of the multi-analyte panel HMHMAG-34K by MILLIPLEX® MAP (Merck, Darmstadt, Germany). The concentration of adiponectin was determined with use of the enzyme-linked immunosorbent assay kit (Cloud-Clone Corp. (CCC, USA), Houston, TX, USA).
The concentrations of TNF-α and IL-6 were determined on the multiplex analyzer MAGPIX (Luminex, USA) using xMAP® Technology with the multi-analyte panel HCYTMAG-60K-PX41 by MILLIPLEX® MAP (Merck, Darmstadt, Germany). The detected information was processed by the special software Luminex xPONENT® with the subsequent export of data to the MILLIPLEX® Analyst 5.1 program.
The multiplex analyzer MAGPIX is based at The Core Facility “Medical genomics” Tomsk National Research Medical Center.
The concentrations of TNF-α and IL-6 were determined on the multiplex analyzer MAGPIX (Luminex, USA) using xMAP® Technology with the multi-analyte panel HCYTMAG-60K-PX41 by MILLIPLEX® MAP (Merck, Darmstadt, Germany). The detected information was processed by the special software Luminex xPONENT® with the subsequent export of data to the MILLIPLEX® Analyst 5.1 program.
The multiplex analyzer MAGPIX is based at The Core Facility “Medical genomics” Tomsk National Research Medical Center.
10
Multiplexed Cytokine Profiling with Luminex
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The Luminex®-based multiplexed immunoassays with fluorescent microspheres Milliplex Map assays (HCYTMAG-60K-PX41, HCYP3MAG-63K-11 and HCP2MAG-62K-PX23, Merck Millipore) were used to measure 72 cytokines/chemokines (five were not detectable).
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