Size-sorted MSCs were seeded in T75 flasks for overnight adherence before media removal the next day. The cells were washed with PBS, and cultured in serum-free media for 48 h at 37 °C, with a 5% CO2 concentration. The media was then collected and centrifuged at 4500g for 15 min to remove cell debris. The supernatant was collected and concentrated to 500 µL with an Amicon Ultra 15 filter (3 kDa cut-off membrane) and stored at −80 °C before analysis. Cell number in each condition was determined to normalize the concentration values of the analytes of interest (MCP-1, IL-6, IL-8, and TGF-β1) obtained using Luminex-based multiplex assays (R&D Systems) according to manufacturer’s instructions. All samples were analyzed in technical duplicates and performed for 2 different donors. The data were obtained with a MAGPIX reader (Millipore), and concentrations were derived from measured mean fluorescence intensities (MFI) using fitted standard curves using 5-parameter logistic regression (SSL5) using the Milliplex Analyst software (Millipore).
Magpix reader
Manufactured by Merck Group
Sourced in United States
The MAGPIX reader is a multiplex detection system that utilizes magnetic microspheres to enable simultaneous measurement of multiple analytes in a single sample. The device employs a combination of magnetic separation and flow cytometry principles to detect and quantify targeted molecules.
Automatically generated - may contain errors
6 protocols using magpix reader
1
Analyzing Secreted Factors from Size-Sorted MSCs
2
Cytokine and Chemokine Profiling in Melanoma
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Blood samples were collected from treated and control mice from both SC and IV model of melanoma at mice sacrifice. Serum samples were analyzed for cytokine and chemokine levels by Mouse cytokine/chemokine magnetic bead panel, Milliplex Map kit (MCYTOMAG-70K-PX32, Millipore, Billerica, MA, USA) accordingly to manufacturer procedures, using a Luminex MagPix reader with xPONENT software (Millipore). Quality controls were included to qualify assay performance and the concentration values respected their ranges.
3
Multiplex Cytokine Quantification
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The concentration of cytokines, including IFN-ɣ and IL-17A, was determined by Luminex®-based multiplex assays (R&D Systems) according to manufacturer’s instructions. All samples were analysed in duplicate and repeated for 6 different donors. The data were obtained with a MAGPIX reader (Millipore), and concentrations were derived from measured mean fluorescence intensities using fitted standard curves using 5-parameter logistic regression (SSL5) using the Milliplex Analyst software (Millipore).
4
Malaria Antibody Quantification using MAP
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IgG to malaria antigens was measured using MAP assay as described before [37 (link)]. Modification includes the use of magnetic plate separator instead of vacuum manifold (Luminex, Austin, Texas, Cat# CN-0269-01) and a MAGPIX reader instead of Luminex 100 instrument (EMD Millipore, Billerica, MA). The results were expressed as median fluorescence intensity (MFI). Negative and positive controls were included on each plate consisting of (a) plasma pool of 3 North Americans who had never travelled to malaria endemic areas as negative control and (b) pool of plasma from 7 Cameroonian multigravidae with high Ab levels to FV2 as positive control.
5
Cytokine Secretion Assay for Autoimmune Cells
6
Multiplex Immunoassay for Malaria Antibodies
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Antigens were coupled to MagPlex-C microspheres (Luminex, Austin, TX) as described previously, with 1 million microspheres coupled with 1 µg of MSP-1, AMA-1, and TTc and 15 µg of CSP [36 (link)]. Immunoglobulin G was measured in a multianalyte platform assay as previously described [30 (link), 36 (link)]. In brief, 50 µL (2000 microspheres/test) antigen-coupled microspheres were incubated with 50 µL maternal and cord plasma at 1:100 dilution in phosphate-buffered saline (PBS) and 1% bovine serum albumin (BSA) for 1 hour at 25°C on a shaker. After 3 washes, microspheres were incubated with 100 µL secondary antibody diluted to 2 µg/mL in PBS-1% BSA per well (R-phycoerythrin-conjugated, Affini Pure F(ab′)2 fragment, goat anti-human IgG Fc fragment specific; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 hour. Microspheres were washed 3 times, resuspended in 100 µL PBS-1% BSA, and analyzed using a MAGPIX reader (EMD Millipore, Billerica, MA). The results were expressed as median fluorescence intensity (MFI). Positive and negative controls were included on each plate consisting of (1) pool of plasma from 6 Cameroonian adults (positive assay control) and (2) pool of plasma from 13 North Americans (negative assay control). In addition, plasma from 20 North American adults was used to determine the cutoff for antibody positivity.
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