A Bio‐plex assay (Bio‐Plex Pro™ Human Cytokine 27‐plex Assay #M500KCAF0Y) from Bio‐Rad (Hercules, CA, USA) was performed to quantify 27 inflammatory cytokines secreted in the supernatant of IBM and CTL fibroblasts after 96 h in culture (undiluted samples) normalized by cell number.
Bio plex pro human cytokine 27 plex assay m500kcaf0y
Manufactured by Bio-Rad
Sourced in United States
The Bio-Plex Pro Human Cytokine 27-plex Assay #M500KCAF0Y is a multiplex assay that can simultaneously measure the concentration of 27 different human cytokines in a single sample. It is designed for use with the Bio-Plex suspension array system.
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4 protocols using bio plex pro human cytokine 27 plex assay m500kcaf0y
1
Cytokine Profiling of IBM and CTL Fibroblasts
2
Cytokine/Chemokine Profiling in Cell Culture
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The concentration of cytokines/chemokines was assessed in cell culture supernatants 72 hpi by using immunoassays formatted on magnetic beads (Bio-Plex Pro Human Cytokine 27-plex Assay #M500KCAF0Y) (Bio-Rad, Hercules, CA, USA), according to manufacturer’s protocol via Luminex 100 technology (Luminex, Austin, TX, USA). A total of 0 pg/mL is attributed to values below the limit of detection.
3
Cytokine/Chemokine Profiling of Infected Cells
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Concentration of cytokines/chemokines was assessed in cell culture supernatants 6 days post infection by using immunoassays formatted on magnetic beads (Bio-Plex Pro Human Cytokine 27-plex Assay #M500KCAF0Y) (Bio-Rad, Hercules, CA, USA), according to manufacturer’s protocol via Luminex 100 technology (Luminex, Austin, TX, USA). Briefly, the capture antibody-coupled beads are first incubated with antigen standards or samples for a specific time. The plate is then washed to remove unbound materials, followed by incubation with biotinylated detection antibodies. After washing away the unbound biotinylated antibodies, the beads are incubated with a reporter streptavidin-phycoerythrin conjugate (SA-PE). Following removal of excess SA-PE, the beads are passed through the array reader, which measures the fluorescence of the bound SA-PE. For the targets over-range an arbitrary value of 4000 pg/mL is assigned, while 0 pg/mL is attributed to values below limit of detection.
4
Quantification of Cytokine Levels
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The collected CMs/growth medium were stabilized using 1% human serum albumin Albunorm (Octapharma AG, Lachen, Switzerland), stored at −80 °C, and analyzed for the cytokine levels using a Bio‐Plex Pro™ Human Cytokine 27‐plex Assay # M50‐0KCAF0Y (Bio‐Rad, Hercules, CA, USA), a Luminex 200™ instrument (Luminex Corporation, Austin, TX, USA), and the bio‐plex manager 6.0 software (Bio‐Rad). Cytokine concentrations were determined from standard curves of known concentrations of recombinant proteins.
Cluster heatmaps of cytokine levels were generated inr (version 3.2.2) using rstudio (version 1.0.136) and the R package Clustermap (Lingjærde, personal communication). In brief, cytokine data were log10‐transformed and median‐centered (only cytokines detected in at least half of the samples were included). Untreated control samples were clustered using Euclidean distance and complete linkage, and the resulting order of cell lines and cytokines was used for visualizing both control and S100A4‐stimulated samples in the heatmaps in Fig. 2A.
Cluster heatmaps of cytokine levels were generated in
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