Conditioned media were collected from every flask in this study (n = 3), centrifuged at 5000 ×g for 5 minutes, and stored at −80°C. Filtrates (50 μL) were analysed using both the Bio-Plex Pro-Human Cytokine 27-Plex and Bio-Plex Pro-Human Cytokine 21-Plex assay (Bio-Rad, USA), according to the manufacturer's instructions (see Supplementary Methods).
Bio plex pro human cytokine 21 plex
Manufactured by Bio-Rad
Sourced in United States
The Bio-Plex Pro Human Cytokine 21-plex is a multiplex immunoassay designed to simultaneously quantify the levels of 21 different human cytokines in a single sample. It is a tool for researchers to measure multiple analytes in a cost-effective and time-efficient manner.
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5 protocols using bio plex pro human cytokine 21 plex
1
Cytokine Profiling of Conditioned Media
2
Cytokine and Protein Profiling in Cervicovaginal Fluid
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The levels of genital cytokines and barrier‐related proteins were assessed in CVL supernatants using multiplexed ELISA assays. Cytokine concentrations were measured using the Bio‐Plex Pro™ Human Cytokine 21‐Plex and 27‐Plex kits (Bio‐Rad Laboratory, Hercules, CA, USA) as previously described (Table S1 ) [28 ]. Concentrations of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were quantified using the MMP 9‐Plex, and TIMP 4‐Plex kits (Bio‐Rad Laboratory), respectively, as instructed by the manufacturer. MMP/TIMP measurements were performed on baseline samples (n = 136; Figure S1 ). All analyte concentrations were measured using the Bio‐Plex® 200 system (Bio‐Rad Laboratory).
3
Multiplex Cytokine and Toxicity Profiling
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A total of 48 analytes were analyzed in serum samples using Bio-Plex multiplex magnetic bead-based antibody detection kits (Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions. In this study, we used the Bio-Plex Pro Human Cytokine 21-plex and Bio-Plex Human Cytokine 27-plex panels. Urine samples were analyzed using the Bio-Rad Human Kidney Toxicity Panel 2 (Bio-Rad, Hercules, CA, USA), which detects albumin, beta-2-microglobulin (β2M), cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), osteopontin, and trefoil factor 3 (TFF3). Serum or urine aliquots of 50 μL were analyzed, and a minimum of 50 beads per analyte were acquired. Each analysis included standards and quality controls. Median fluorescence intensities were measured using a Luminex 100 or 200 analyzer (Luminex, Austin, TX, USA). Each sample was analyzed in triplicate. Standard curves for each cytokine were generated using standards provided by the manufacturer, and data analysis was performed using the MasterPlex CT control software 1.0 and MasterPlex QT analysis software (MiraiBio, Alameda, CA, USA).
4
Multiplexed Serum Analyte Profiling
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Serum levels of 94 analytes was analyzed using Bio-Plex (Bio-Rad, Hercules, CA, USA) multiplex magnetic bead-based antibody detection kits following manufacturer’s instructions. Multiplex kits (Bio-Rad, Hercules, CA, USA), Bio Plex Pro Human Cytokine 21-plex, Bio Plex Human Cytokine 27-plex, Bio Plex Human Cytokine 37-plex, and Bio-Plex Human MMP Panel panels were used in the study. Data collected was analyzed using with MasterPlex CT control software and MasterPlex QT analysis software (MiraiBio, Alameda, CA, USA).
5
Multiplex Cytokine Profiling of Patient Sera
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Serum levels of immune mediators were measured using the Bio-Plex Pro™ human cytokine 21-plex (CTACK, GRO-α, HGF, IFN-α2, IL-12p40, IL-16, IL-18, IL-1α, IL-2Ra, IL-3, LIF, M-CSF, MCP-3, MIF, MIG, SCF, SCGF-β, SDF-1α, TNF-β, TRAIL, β-NGF) and 27-plex (Basic-FGF, Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-10, IL-13, IL-15, IL-17A, IL-1β, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, IL-12p70, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, VEGF) immunoassay kits (Bio-Rad) according to the manufacturer’s instruction. Briefly, magnetic beads were aliquoted in 96-well plates followed with addition of standards and sera from patients and control subjects. After an incubation period, plates were washed using a magnetic wash station according to manufacturer’s instructions, followed with addition of a detection antibody. Plates were incubated for a further 30 minutes and washed, followed with a final incubation of 10 minutes in the presence of streptavidin-PE. Results were acquired using the flexMAP™3D (Luminex corp.) with Luminex xPONENT® software, based on standard curves plotted through a 5-parameter logistic curve setting.
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