biotinylated goat anti human cd127 polyclonal ab  (R&D Systems)

Journal: Nature communications

Article Title: Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele.

doi: 10.1038/s41467-019-12393-1

Figure Lengend Snippet: Fig. 4 Monocyte surface IL7R is functional, activating multiple transcriptional pathways. a Violin plot demonstrating monocytes from PBMC cultures in the untreated state, after exposure to LPS and after exposure to LPS with recombinant IL-7. IL-7 leads to marked downregulation of IL7R on LPS stimulated monocytes, indicative sensitivity to exogenous cytokine (paired t-test). b Volcano plot of mRNA from RNAseq experiments of 8 paired monocyte samples either treated with LPS alone for 24 h or with LPS with additional IL-7 added for the last 2 h of culture. Treatment leads to widespread differential transcript expression with the most significant 20 transcripts labelled. c Example boxplots of genes differentially regulated by recombinant IL-7 in monocytes (linear model). d t-SNE plot of single-cell real-time quantitative PCR (RT-qPCR) data from 10 individuals (1218 cells) either untreated or in the presence LPS showing expression of membrane-bound IL7R (IL7Rmb), soluble IL7R (sIL7R), CLEC4E, TNF, CUX1, AKT1, STAB1, CD163, CSF1R, CCL5, DDX39A, DDX39B, MALAT1, LTB, IFNB1, CD14, FCGR3B, LYZ, and ACTB. e Density plots of single-cell RT-qPCR expression of sIL7R and mbIL7R normalized expression in untreated and LPS treated monocytes according to rs6897932 genotype. Kolmogorov Smirnov test. f Violin plots of single-cell RT-qPCR expression of MALAT1 in LPS stimulated monocytes either alone or in the presence of recombinant human IL-7 according to rs6897932 genotype, t-test

Article Snippet: Plates were blocked with 5% BSA for 1 h, washed, and cell supernatants added for 2 h. Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Functional Assay, Recombinant, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Membrane

Journal: Nature communications

Article Title: Context-specific regulation of surface and soluble IL7R expression by an autoimmune risk allele.

doi: 10.1038/s41467-019-12393-1

Figure Lengend Snippet: Fig. 5 IL7R+ monocytes form a distinct subset detectable in synovial fluid. a Representative flow cytometry of IL7R staining of spondyloarthritis matched patient PBMC (left) and SFMC (middle isotype control, right) gated on CD3-CD14+ as per sequential gating strategy shown in Supplementary Fig. 1. b Results from 4 patients demonstrating comparative monocyte IL7R staining in matched PBMC and SFMC using flow cytometry. c t-SNE clustering of monocytes from single-cell RNA sequencing of spondyloarthritis patient SFMC showing 5 clusters (n = 3). d Single-cell monocyte expression of IL7R, LTB, CD14, CCL5, IL32, and TCF7 overlaid on t-SNE clustering in c. e Volcano plot of genes significantly upregulated in the SFMC monocyte cluster 4 compared to all other SFMC monocyte clusters with top 20 genes annotated. Wilcoxon rank sum test. f Heatmap of top 10 genes from the each of the five SFMC monocyte clusters identified in c

Article Snippet: Plates were blocked with 5% BSA for 1 h, washed, and cell supernatants added for 2 h. Bound sIL7R was detected with 12.5 ng/ml biotinylated goat anti-human CD127 polyclonal Ab (R&D Systems) for 1 h, followed by a 30 min incubation with streptavidin-HRP and a 15 min incubation with TMB peroxidase substrate (Thermo Fisher).

Techniques: Cytometry, Staining, Control, RNA Sequencing, Expressing