Mice were anesthetized via i.p. administration of ketamine/xylazine and placed on a stereotactic frame. Cerebrospinal fluid was collected from the cisterna magna under a dissection microscope using a glass capillary (Sutter Instrument, B100–50–10, pulled with a Sutter Instrument P-30 micropipette puller to a size of 0.5 mm in diameter). CSF (12.5 μl ) was obtained from each mouse and analyte quantification performed using Luminex magnetic beads with the Bio-Plex Pro Mouse Cytokine Panel 23-plex instruction (Bio-Rad). Data were acquired with the Luminex Flexmap 3D and analyzed with xPONENT Software V4.2 (both from Luminex Corp., Austin, TX).
Xponent software v4
Manufactured by DiaSorin
Sourced in United States
XPONENT Software V4.2 is a laboratory equipment software developed by DiaSorin. It provides a platform for data management and analysis related to diagnostic testing.
Automatically generated - may contain errors
Lab products found in correlation
Flexmap 3d, by DiaSorin (4 mentions)
B100 50 10, by Sutter Instruments (2 mentions)
P 30 micropipette puller, by Sutter Instruments (2 mentions)
Bio plex pro mouse cytokine panel 23 plex instruction, by Bio-Rad (2 mentions)
Milliplex map human high sensitivity t cell magnetic bead panel, by Merck Group (1 mentions)
Milliplex map porcine cytokine chemokine magnetic bead panel, by Merck Group (1 mentions)
Sector imager 6000, by Mesoscale (1 mentions)
Flexmap 3d instrument, by DiaSorin (1 mentions)
Luminex 200, by DiaSorin (1 mentions)
Luminex 200 detection instrument, by DiaSorin (1 mentions)
Hcyta 60k, by Merck Group (1 mentions)
8 protocols using xponent software v4
1
Cerebrospinal Fluid Collection and Cytokine Analysis
2
Multiplex Immunoassay for IL-4 and IL-21 Analysis
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Determinations of IL-4 and IL-21 were performed using a multiplex bead-based immunoassay (MILLIPLEX® MAP human high-sensitivity T cell magnetic bead panel; Merck EMD Millipore, Billerica, MA) following the manufacturer’s instructions. The assay was performed on the Luminex® MAGPIX® System detection instrument operated with xPONENT Software V4.2 (both from Luminex Corp., Austin, TX). The results were analyzed using the Belysa™ Analysis software V1.2.0 (Merck KGaA, Darmstadt, Germany).
3
Porcine Cytokine Quantification Protocol
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Blood samples were allowed to coagulate in their vacutainer tubes for approximately 10–15 min before being centrifugated at 1,000 × g for 10 min at 4 °C. The serum was aliquoted, transported to the laboratory and stored at − 20 °C37 (link). The concentrations of porcine cytokines interleukin 1 alpha (IL-1α), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrotic factor alpha (TNFα), and interleukin 10 (IL-10) were individually measured according to the manufacturer’s instructions using the MILLIPLEX MAP Porcine cytokine/chemokine magnetic bead panel (Millipore) in the MAGPIX system with xPONENT software V4.2 (Luminex corporation)22 (link). The processes were repeated twice, and the cytokine concentrations were determined as ng/mL unit.
4
Multiplex Cytokine Analysis of CSF Samples
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CSF samples were stored frozen after the initial series of routine laboratory tests were performed. Cytokines in the CSF from cases 12 and 13 were analyzed by a bead-based multiplex fluorescence assay [15 (link)]. The Procartaplex Human Cytokine Magnetic 35-plex Bead Panel (Product #LHC6005M, Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) simultaneously evaluates 35 analytes (EGF, Eotaxin, FGF-basic, G-CSF, GM-CSF, HGF, IFNa, IFNγ, IL-1a, IL-1β, IL-1RA, IL-2, IL-2R, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-17A, IL-17F, IL-22, IP-10, MIG, MIP-1α, MIP-1β, MCP-1, RANTES, TNFα, and VEGF). This assay was run on the Luminex® FLEXMAP 3D® instrument operated with xPONENT Software V4.2 (both from Luminex Corp., Austin, TX, USA). The cytokine profiling was carried out at the Bursky Center for Human Immunology and Immunotherapy Programs at the Washington University Immunomonitoring Laboratory, St. Louis, MO, USA.
5
Analyte Quantification in Microtiter Plates
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Analyte levels in the LMX microtiter plates were assessed using a FLEXMAP® 3D instrument operated with xPONENT Software V4.2 (both from Luminex), and MSD plates were read on a SECTOR® Imager 6000 operated with MSD Discovery workbench software v.3.0/4.0 (both from Meso Scale Diagnostics).
6
Multiplex Protein Analysis with xMAP
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The assay was run on the LED/image-based MagPix, and laser/flow-based Luminex 200 and FlexMap 3D xMAP instruments operated with xPONENT Software V4.2 (MagPix and FlexMap 3D) and V4.3 (Luminex 200) (all from Luminex Corp.) [23 ]. The classic 200 unit is designed for multiplex analysis up to 100 analytes in a single sample, and reads 96-well microtiter plates in ≈ 45 min. The MagPix instrument is more compact and portable than the 200 model to accommodate settings with space constraints or fieldwork, and simultaneously measures 50 analytes in 96-well plates in ≈ 60 min. Both the 200 and MagPix models provide single-digit picogram/mL sensitivity for protein targets and ≥ 3.5 logs of dynamic range. The newer FlexMap 3D allows evaluation of up to 500 analytes in a single sample. It has increased sensitivity (sub-picogram/mL) and dynamic range (≥ 4.5 logs) compared to earlier instruments, and accommodates high-throughput analysis and more advanced automation. The FlexMap 3D reads 96-well plates in ≈ 20 min and 384-well plates in ≈75 min.
7
Cerebrospinal Fluid Collection and Cytokine Analysis
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Mice were anesthetized via i.p. administration of ketamine/xylazine and placed on a stereotactic frame. Cerebrospinal fluid was collected from the cisterna magna under a dissection microscope using a glass capillary (Sutter Instrument, B100–50–10, pulled with a Sutter Instrument P-30 micropipette puller to a size of 0.5 mm in diameter). CSF (12.5 μl ) was obtained from each mouse and analyte quantification performed using Luminex magnetic beads with the Bio-Plex Pro Mouse Cytokine Panel 23-plex instruction (Bio-Rad). Data were acquired with the Luminex Flexmap 3D and analyzed with xPONENT Software V4.2 (both from Luminex Corp., Austin, TX).
8
Multiplex Cytokine Profiling of Plasma
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Plasma samples were freshly thawed, and each sample was analyzed for a panel of 39 cytokines by Luminex bead-based multiplex assay using the MILLIPLEX ® MAP Human Cytokine/Chemokine Magnetic Panel II (#HCYP2MAG-62K; Merck EMD Millipore, Billerica, MA, USA) with analytes IL-33, IL-21, and TRAIL and Human Cytokine/Chemokine/Growth Factor Magnetic Panel A (#HCYTA-60K; Merck EMD Millipore, Billerica, MA, USA) with analytes sCD40L, EGF, eotaxin, FGF-2, FLT-3L, fractalkine, G-CSF, GM-CSF, GRO-α, IFNα2, IFNɣ, IL-1α, IL-1β, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17E, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, TGFα, TNFα, and VEGF-A. The plasma samples were diluted, and the assay was performed using the manufacturer’s protocol.
Standard curves for each cytokine were generated using the premixed lyophilized standards provided in the kit. Each sample was run in duplicate, and an average of the duplicates was used as the measured concentration. Both assays were run on the Luminex 200 detection instrument operated with xPONENT Software V4.2 (Luminex Corp., Austin, TX, USA).
Standard curves for each cytokine were generated using the premixed lyophilized standards provided in the kit. Each sample was run in duplicate, and an average of the duplicates was used as the measured concentration. Both assays were run on the Luminex 200 detection instrument operated with xPONENT Software V4.2 (Luminex Corp., Austin, TX, USA).
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