We used 50 μL of adult bone marrow CECs’ (n = 6) and fetal liver parenchyma CECs’ (n = 6) culture media to quantify culture medium cytokines in doubles using a Bio-Plex Pro Human Cytokine Screening Panel, 48-Plex (BioRad, #12007283, Hercules, CA, USA) and a Bio-Plex 200 instrument (BioRad, Hercules, CA, USA).
Bio plex pro human cytokine screening panel 48 plex
Manufactured by Bio-Rad
Sourced in United States
The Bio-Plex Pro Human Cytokine Screening Panel 48-plex is a multiplexed assay designed to quantify the levels of 48 different cytokines and chemokines in biological samples. The panel uses magnetic bead-based technology to enable the simultaneous measurement of multiple analytes in a single well, providing a comprehensive analysis of the cytokine profile.
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Lab products found in correlation
Bio plex cell lysis kit, by Bio-Rad (5 mentions)
Sample diluent, by Bio-Rad (3 mentions)
Bio plex pro human cytokine icam 1, by Bio-Rad (3 mentions)
Bio plex pro human cytokine vcam 1, by Bio-Rad (3 mentions)
Bio plex 200 system, by Bio-Rad (3 mentions)
Magpix analyzer, by DiaSorin (3 mentions)
Bio plex 100 system, by Bio-Rad (3 mentions)
Laser microdissection v5, by Leica camera (2 mentions)
Bio plex manager, by Bio-Rad (2 mentions)
Bio plex pro human cytokine panel 14 plex and pro human th17 3 plex set, by Bio-Rad (2 mentions)
Bio plex, by Bio-Rad (2 mentions)
Bio plex manager 6, by Bio-Rad (2 mentions)
Bio plex 200 instrument, by Bio-Rad (1 mentions)
Manager 4, by Bio-Rad (1 mentions)
Bio plex manager software 6, by Bio-Rad (1 mentions)
Cytotox 96 non radioactive cytotoxicity assay, by Promega (1 mentions)
Antibody isotyping 7 plex human procartaplex panel, by Thermo Fisher Scientific (1 mentions)
Pro washer station, by Bio-Rad (1 mentions)
3d instrument, by Bio-Rad (1 mentions)
Bio plex magpix multiplex reader, by Bio-Rad (1 mentions)
32 protocols using bio plex pro human cytokine screening panel 48 plex
1
Cytokine Profiling of Bone Marrow and Fetal Liver CECs
2
Multiplex Analysis of Cryopreserved PDA Tissue
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Cryopreserved PDA tissue samples were cut into multiple consecutive sections (20 µm) and then stained with cresyl-violet. Areas of tumor epithelium and stroma were highlighted using brightfield microscopy at 20× magnification. Laser microdissection was performed with the Leica Laser Microdissection V5.0.2.0 software. Sufficient protein concentrations for multiplex analysis could be obtained by the dissection of 30–50 × 106 mm2. The tissue was lysed using the BioPlex™ Cell Lysis Kit (Bio-Plex Cell Lysis Kit, BioRad, Hercules, CA, USA; 171304011) according to the manufacturer´s instructions. Serum samples were thawed overnight at 4 °C and then diluted at 1:1 using Sample Diluent (BioRad, Hercules, CA, USA) prior to protein quantification. A two-laser array reader simultaneously quantified all proteins of interest. The concentrations were calculated using Bio-Plex Manager 4.1.1 and a 5-parameter logistic plot regression formula. The Bio-Plex Pro Human Cytokine Screening Panel 48-plex (BioRad, Hercules, CA, USA; 12007283), Bio-Plex Pro Human Cytokine ICAM-1 (BioRad, Hercules, CA, USA; 171B6009M) and Bio-Plex Pro Human Cytokine VCAM-1 (BioRad, Hercules, CA, USA; 171B6022M) were used for the quantification of immunological parameters.
3
Multiplex Cytokine Quantification
4
Epithelial Cytokine Quantification in Airway Samples
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Epithelial cytokines present in apical washes were assessed by multiplex assay using the Bio‐Plex Pro Human Cytokine Screening Panel 48‐Plex (Bio‐Rad, Hercules, CA, USA), with plates read on the Bio‐Plex 200 System (Bio‐Rad) and results calculated on Bio‐Plex Manager Software 6.0 (Bio‐Rad). IL‐8 was assessed by ELISA, using the BD OptEIA Set for human IL‐8 (BD Biosciences, Franklin Lakes, NJ, USA) as previously described.17 , 41 Endotoxin levels in ALI washes were measured using the KCA‐Endochrome‐K (Charles River, Wilmington, MA, USA) Limulus amoebocyte lysate kinetic chromogenic assay according to manufacturer’s instructions. Lactate dehydrogenase was measured using the CytoTox 96 Non‐Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA).
5
Multiplex Immunoassay for Plasma Proteins
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The concentration of fibrinogen in the plasma was determined with AssayMax Human Fibrinogen ELISA Kit (Assaypro, St. Charles, MO, USA). The concentration of immunoglobulin in the serum was determined using Antibody Isotyping 7‐Plex Human ProcartaPlex Panel (Invitrogen) according to the manufacturer's instruction. The other cytokines in the serum were measured using Bio‐Plex Pro Human Cytokine Screening Panel (48‐Plex; Bio‐Rad, Hercules, CA, USA) according to the manufacturer's instruction. The cytokines included in the analysis are those for which more than 70% of the tested samples showed values within their standard range.
6
Cytokine and Chemokine Profiling Protocol
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Cytokine and chemokine levels were determined using the magnetic bead suspension array using Bio-Plex Pro™ Human Cytokine Screening Panel (48 plex) and Human Chemokine Panel (40 plex) following the manufacturer’s directions (Bio-Rad Laboratories, Hercules, CA, USA). Fifty µL of cell-free culture medium was analyzed by collecting a minimum of 50 beads per analyte. Median fluorescence intensities were collected using a MAGPIX analyzer (Luminex, Austin, TX, USA). Each sample was analyzed in triplicate. Data collected were analyzed with MasterPlex CT control software and MasterPlex QT analysis software (MiraiBio, San Bruno, CA, USA). Standard curves for each cytokine were generated using standards provided by the manufacturer. The average values of measured cytokine and chemokine levels were presented as a heatmap using the web-based tool Heatmapper (http://www.heatmapper.ca/ (accessed on 7 December 2022)) [109 (link)].
7
Multiplex Cytokine Profiling Using Bio-Plex Pro
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The Bio-Plex Pro Human Cytokine Screening Panel, 48-Plex (#12007283), was purchased from Bio-Rad and used according to the manufacturer’s instructions. Beads were added to a 96-well plate and washed in a Bio-Plex Pro Washer Station. Samples were added to the plate containing the mixed antibody-linked beads and incubated for 1 h at room temperature. Room temperature incubation steps were performed on an orbital shaker at 500–600 rpm. Following the incubation, plates were washed in a Bio-Plex Pro Washer Station and incubated with a biotinylated detection antibody for 30 min at room temperature under shaking conditions. The plates were washed as described above, and streptavidin-PE was added. After incubation for 10 min at room temperature, a wash was performed as described previously, and assay buffer was added to the wells. Each sample was assayed in singlets. Plates were read on a Bio-Plex 3D instrument with a lower bound of 50 beads per sample per cytokine/chemokine. Data were analyzed with Luminex xPONENT for FLEXMAP3D software v.4.3 (Luminex).
8
Protein Profiling of Pancreatic Tumor Microenvironment
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First, serial sections (20 µm) of cryopreserved PDA tissue samples were stained with cresyl-violet. Then, Laser Capture Microdissection was performed with the Leica Laser Microdissection V5.0.2.0 software to separate the stromal compartment from tumor epithelium. The procedure was performed according to the protocol of the inventors (11 (link)). Both, the stromal tissue as well as the 3D bioprints were lysated using the BioPlex™ Cell Lysis Kit (Bio-Plex Cell Lysis Kit, BioRad, USA; 171304011) according to manufacturer´s instructions. Serum samples were thawed overnight at 4°C and diluted 1:1 prior to protein quantification (Sample Diluent, BioRad, USA). All proteins of interest were concurrently quantified using a two-laser array reader. Concentrations were calculated with Bio-Plex Manager 4.1.1 based on a 5-parameter logistic plot regression formula. Bio-Plex Pro Human Cytokine Screening Panel 48-plex (BioRad, USA; 12007283), Bio-Plex Pro Human Cytokine ICAM-1 (BioRad, USA; 171B6009M) and Bio-Plex Pro Human Cytokine VCAM-1 (BioRad, USA; 171B6022M) were utilized to quantify cytokine concentrations.
9
Multiplex Cytokine Profiling for Vaccine Monitoring
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To monitor abnormal and prolonged cytokine production, the cytokine levels in serum samples 1–2 weeks after the second and third vaccination doses were analyzed using the Bio-Plex Pro Human Cytokine Screening Panel 48-plex (#12007283) (Bio-Rad) according to the manufacturer’s instructions (Figures S3 and S4 ). Briefly, 50 μL of the serum sample was mixed with capture antibody-coupled beads in a 96-well plate and incubated on a shaker at 850 rpm for 1 h at room temperature. The beads were washed, mixed with 50 μL of biotinylated detection antibodies, and incubated on a shaker at 850 rpm for 30 min at room temperature. After washing, 50 μL of streptavidin-phycoerythrin reporter dye was added and incubated on a shaker at 850 rpm for 10 min at room temperature. The beads were washed and resuspended in 125 μL assay buffer. Fluorescence intensities were measured using a Bio-Plex MAGPIX multiplex reader (Bio-Rad) and the data were analyzed using Bio-Plex Manager Software version 6.0.
10
Multiplex Immunoassay Profiling of EVs
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Immune molecules in plasma were previously measured using a magnetic bead-based 61-plex immunoassay (customized ProcartaTM immunoassay, Affymetrix) [26 (link)]. The immune profiling of extracellular vesicles was performed at the Human Nutritional Chemistry Service Laboratory at Cornell University using a human 48-plex magnetic bead kit (Bio-Plex Pro Human Cytokine Screening Panel, 48-plex, Bio-Rad). Prior to analysis, EV samples were treated with Triton 1% to allow the release of encapsulated cytokines [32 (link)]. Each sample was measured in duplicate on a MAGPIX® Multiplexing System (Luminex Corp.). For each well, we used the median fluorescence intensity of all beads measured for a given analyte and averaged the two replicates and results were accepted when the coefficient of variation (CV) was below 15%.
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