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.
Hcyp2mag 62k
Manufactured by Merck Group
HCYP2MAG-62K is a laboratory equipment product from Merck Group. It is a magnetic separation system designed for high-throughput biomolecule purification. The core function of this product is to facilitate the efficient separation and isolation of target biomolecules from complex samples using magnetic beads.
Automatically generated - may contain errors
Lab products found in correlation
Hcytomag 60k, by Merck Group (3 mentions)
Hcytmag 60k px41, by Merck Group (2 mentions)
Human scd40l platinum elisa kit, by Thermo Fisher Scientific (1 mentions)
Luminex magpix system 40 072, by Merck Group (1 mentions)
Milliplex analyst, by Merck Group (1 mentions)
Magpix, by Merck Group (1 mentions)
4 protocols using hcyp2mag 62k
1
Multivariate cytokine profiling in BAL and plasma
2
Multiplex Cytokine Analysis in Serum and SF
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Serum or SF samples (25 μL) from all 21 patients were analyzed using 3 kits: HCYTOMAG-60K (IFNγ, IL-10, IL-17A, IL-1β, IL-4, IL-6, IL-8, CXCL10, and TNFα), HCYP3MAG-63K (CXCL9), and HCYP2MAG-62K (CXCL13, IL-23, and IL-33) (Millipore). The samples and standards were analyzed using Luminex 200 following the manufacturer's instructions. All samples from each kit were analyzed on the same plate.
3
Comprehensive Plasma Profiling for Biomarker Discovery
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After donation, blood samples were immediately spun at 2500 rpm for 15 minutes at room temperature, and 2-ml plasma aliquots with 40 μl of dipeptidyl peptidase-4 protease inhibitor (Merck Millipore, Billerica, MA, USA) were stored at −80 °C. All plasma samples were thawed only once for determination of the concentration of 13 soluble receptors (HSCR-32 K; Merck Millipore) and 64 cytokines (HCYTOMAG-60 K and HCYP2MAG-62 K; Merck Millipore) by addressable laser bead immunoenzyme assay according to the manufacturer’s specifications and read using the Luminex MAGPIX® System 40-072 (Merck Millipore). sCD40L plasma levels were subsequently assessed by enzyme-linked immunosorbent assay (ELISA) using the Human sCD40L Platinum ELISA kit (eBioscience, San Diego, CA, USA) according to the manufacturer’s specifications and read at λ = 450/570 nm in a VICTOR X3 2030 multilabel ELISA reader (PerkinElmer, Singapore).
4
Cytokine and Chemokine Profiling of Cells
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Cytokine and chemokine responses of PBMCs, SkMCs, HMEC-1, and HFFs were measured using methods previously reported by us19 (link),36 (link),37 (link) with the following Milliplex kits: HCYTMAG-60K-PX41, HCYPMAG-63K, and HCYP2MAG-62K (EMD Millipore, Billerica MA). Cells were seeded at a density of 105 cells per well. Cells were either stimulated for 16 h with graded concentrations of TLR ligands, or mock-stimulated with vehicle (DMSO). Supernatants were collected by Precision 2000 liquid handlers (BioTek, Winooski, VT) and diluted 1:3 for each kit. The data was acquired on a MagPix® instrument and the data analyzed using Milliplex Analyst (EMD Millipore, Billerica MA).
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