goat anti human il 18rα il 1r5 neutralizing antibody  (R&D Systems)

Journal: iScience

Article Title: Phenotypic and functional characterization of pharmacologically expanded Vγ9Vδ2 T cells in pigtail macaques

doi: 10.1016/j.isci.2023.106269

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Article Snippet: Mouse monoclonal anti-CD218 (IL-18Rα) PE-Vio770 conjugated (clone: H44) , Miltenyi Biotec , Cat# 130-101-723; RRID: AB_2656352.

Techniques: Recombinant, Staining, Random Hexamer, Conjugation Assay, Reverse Transcription, Software, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Histamine Activates Human Eosinophils via H 2 R and H 4 R Predominantly in Atopic Dermatitis Patients

doi: 10.3390/ijms231810294

Figure Lengend Snippet: IL-18Rα expression in eosinophils compared between AD patients and healthy controls after stimulation with histamine, ST-1006, and amthamine. ( a – d ) Eosinophils from AD patients (black) and healthy controls (orange) were stimulated with histamine (10 µmol/L), the H 4 R agonist ST-1006 (10 µmol/L), and the H 2 R agonist amthamine (10 µmol/L) for 6 h ( a , b ) or 24 h ( c , d ) or were left unstimulated (NS). The expression level of IL-18Rα was measured at mRNA level by real-time PCR. ( e ) The constitutional IL-18Rα mRNA expression of non-stimulated (NS) purified eosinophils from AD-patients (black) and healthy controls (orange) is shown. ( f ) Eosinophils were stimulated with histamine (10 µmol/L) for 20 h and IL-18Rα expression was measured by flow cytometry. The extent of histamine-induced IL-18Rα expression (n-fold induction of IL-18Rα protein expression) was correlated with the number of eosinophils in the blood of each subject for statistical analysis, a Mann–Whitney test ( e ), a Spearman correlation ( f ), and a Wilcoxon signed rank test ( a – d ) were applied. * p -value < 0.05; ** p -value < 0.1. NS—not stimulated, AD—atopic dermatitis.

Article Snippet: Cultured eosinophils were harvested after 20 h of incubation since experience has shown that we detect a robust effect in protein expression of histamine receptor stimulation after approximately 18–24 h. Subsequently, we stained with the PE Mouse Anti-Human CD218a (IL-18Rα) antibody (BD Biosciences, San Jose, CA, USA) and the FITC Mouse Anti-Human CD69 (Biolegend, San Diego, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Purification, Flow Cytometry, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Histamine Activates Human Eosinophils via H 2 R and H 4 R Predominantly in Atopic Dermatitis Patients

doi: 10.3390/ijms231810294

Figure Lengend Snippet: IL-18Rα protein expression upon stimulation with histamine, ST-1006, and amthamine. The expression level of the IL-18Rα on purified eosinophils was measured by flow cytometry. ( a ) Eosinophils from one representative donor were gated based on forward scatter (FSC) and side scatter (SSC) (A). Dead cells were labeled with the dye 7-AAD and excluded from the analysis by gate B. Gate C for IL-18Rα positive cells was set based on the isotype control. ( b , c ) Eosinophils from AD patients (black) and healthy controls (orange) were stimulated with histamine (10 µmol/L), the H 4 R agonist ST-1006 (10 µmol/L), or the H 2 R agonist amthamine (10 µmol/L) for 20 h or left unstimulated (NS). ( d , e ) For blocking experiments, the H 4 R antagonist JNJ7777120 (10 µmol/L) and the H 2 R antagonist ranitidine (10 µmol/L) were added 30 min before histamine. For statistical analysis, a Wilcoxon signed rank test was applied. * p -value < 0.05; ** p -value < 0.1. NS—not stimulated.

Article Snippet: Cultured eosinophils were harvested after 20 h of incubation since experience has shown that we detect a robust effect in protein expression of histamine receptor stimulation after approximately 18–24 h. Subsequently, we stained with the PE Mouse Anti-Human CD218a (IL-18Rα) antibody (BD Biosciences, San Jose, CA, USA) and the FITC Mouse Anti-Human CD69 (Biolegend, San Diego, CA, USA).

Techniques: Expressing, Purification, Flow Cytometry, Labeling, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Histamine Activates Human Eosinophils via H 2 R and H 4 R Predominantly in Atopic Dermatitis Patients

doi: 10.3390/ijms231810294

Figure Lengend Snippet: Histamine potentiates the IFN-γ-mediated IL-18Rα protein induction via H 2 R and abolishes the IL-5-mediated downregulation via H 4 R in eosinophils. The expression level of the IL-18Rα was measured by flow cytometry. ( a , b ) Purified eosinophils from AD patients (black) and healthy controls (orange) were stimulated with histamine (10 µmol/L), INF-γ (10 ng/mL), or the combination of both stimuli for 20 h. The H 2 R antagonist ranitidine (10 µmol/L) was added 30 min before histamine. ( c , d ) Purified eosinophils from AD patients (black) and healthy controls (orange) were stimulated with histamine (10 µmol/L), IL-5 (10 ng/mL), or the combination of IL-5 with histamine and H 4 R agonist ST-1006 (10 µmol/L), respectively, for 20 h. For statistical analysis, a Wilcoxon signed rank test was applied. * p -value < 0.05; ** p -value < 0.1; *** p -value < 0.01; NS—not stimulated.

Article Snippet: Cultured eosinophils were harvested after 20 h of incubation since experience has shown that we detect a robust effect in protein expression of histamine receptor stimulation after approximately 18–24 h. Subsequently, we stained with the PE Mouse Anti-Human CD218a (IL-18Rα) antibody (BD Biosciences, San Jose, CA, USA) and the FITC Mouse Anti-Human CD69 (Biolegend, San Diego, CA, USA).

Techniques: Expressing, Flow Cytometry, Purification

Journal: Immunity

Article Title: In situ maturation and tissue adaptation of type 2 innate lymphoid cell progenitors

doi: 10.1016/j.immuni.2020.09.002

Figure Lengend Snippet: (A-C), t-SNE maps of neonatal lung ILC single-cell transcriptomes highlighting RaceID3 clusters (A) or sorted ST2- and ST2+ ILCs (B), and log2 normalized expression of representative genes marking putative ILC progenitors (C). (D) Expression of candidate genes within neonatal lung clusters with at least 10 cells. Color represents z-score mean expression across clusters and dot size represents fraction of cells in the cluster expressing the gene. (E) FACS analysis of neonatal (P4) and adult (6-8w) Il18r1+ST2- lung ILCs, represented as t-SNE maps, indicating expression levels for Gata3, RORγt, PLZF, Tcf7, C-kit, CD103 and Ki67. (F) Histograms comparing ILC subsets (PLZF+Gata3+, RORγt+CD103-, RORγt+CD103+, Il18r1-ST2- ILC2s and RORγt+ ILC3) in adult lung for Gata3, Icos, a4p7, PD1, CD25 and Il17rb expression levels. Data are representative of 2-3 independent experiments (n=6-12 mice). (G) t-SNE map of Il18r1+Icos+ BM ILCs highlighting RaceID3 clusters. (H) Expression of candidate genes for Il18r1+Icos+ BM clusters with at least 10 cells. Color represents z-score mean expression across clusters and dot size represents fraction of cells in the cluster expressing the gene. (I-J) FACS analysis of human lung and PB ILCs pre-gated on CD45+Lin- CD127+EOMES-T-bet-CRTH2-NKp44-c-KIT+/- (I) or c-KIT ILCs (J). Figures depict representative staining (I) and t-SNE maps (J) indicating the expression levels of selected markers. Data are representative of 3 independent experiments (n=2-4 samples each).

Article Snippet: Anti-human ICOS APC (Clone REA192) , Miltenyi , Cat# 130-101-723.

Techniques: Expressing, Staining

Journal: Immunity

Article Title: In situ maturation and tissue adaptation of type 2 innate lymphoid cell progenitors

doi: 10.1016/j.immuni.2020.09.002

Figure Lengend Snippet: (A-B) FACS analysis of Ki67 expression and intravenous CD45 label (ivCD45) in Il18r1+ST2-RORγt+Klrg1- and Il18r1-ST2+ adult lung ILC2s. Representative gating (A) and percentage of ivCD45+ cells within indicated ILC subsets (B). (C) Number of Lin-Il7ra+Il18r1+ cells per ml blood in PBMCs (Il18r1+ST2- ILCs) and lung (ivCD45+Il18r1+ST2-RORγt+Klrg1- ILCs). Graphs in (B) and (C) depict data as mean ± SD (unpaired t-test, *P < 0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant). (D-E) Representative gating (D) and percentage of Ki67+ and ivCD45+ cells (E) for indicated Il18r1+ST2- ILC subsets in adult lung. Data are pooled from 3-4 independent experiments (n=4-16 mice). (F) Percentage of Il5Cre fate-mapped (Il5FM+) Il18r1+ST2-RORγt-CD103- ILCs in lung and BM. RORγt staining was used to exclude ILC3. Data are pooled from 2-3 independent experiments (n=4-9 mice). Graphs in (E) and (F) depict data as mean ± SD (one-way ANOVA Tukey’s multiple comparisons test (E) or unpaired t-test (b, c, f), ***P < 0.001; ****P < 0.0001). Data are pooled from 3-4 independent experiments with a total of n = 8-15 mice). (G) t-SNE representation of single-cell transcriptomes of lung Il18r1+Icos+ ILCs before (“input”) and after culture (“output”) highlighting RaceID3 clusters. (H) Cluster composition of samples (left) and sample composition of clusters (right, for legend see (G)). (I) Expression of candidate genes for input and output populations. Color represents log2 mean expression in the respective cluster and dot size indicates fraction of cells expressing the gene in the cluster. (J) Total number of Gata3+ ILC2s after culture of indicated lung ILCs for 15 days in the presence of Il2, Il7, SCF, Il25 and Il33 on OP9-DL1 for 15 days. (K) FACS analysis of Lin-Il7ra+ lung ILCs on d21 post transfer into sublethally irradiated CD45.2+RAG-/-γc-/- mice. Congenically marked Il18r1+ST2- and Il18r1-ST2+ ILCs sorted from lungs of d2 Nb infected mice were cotransferred (2,000 cells each). Data are representative of 2 individual experiments (n=3 mice). (L) Total number of Gata3+ ILC2 after culture of indicated lung ILCs as in (J). Data in (J) and (L), are pooled from 2 independent experiments with n=3-4 repeat wells each. (M) Indicated lung and BM ILC subsets were sorted as single cells onto OP9-DL1 monolayers and cultured for 19 days. Clonal progeny was analyzed by FACS. Pie charts indicate distribution of culture output across positive single cell cultures.

Article Snippet: Anti-human ICOS APC (Clone REA192) , Miltenyi , Cat# 130-101-723.

Techniques: Expressing, Staining, Irradiation, Infection, Cell Culture

Journal: Immunity

Article Title: In situ maturation and tissue adaptation of type 2 innate lymphoid cell progenitors

doi: 10.1016/j.immuni.2020.09.002

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Article Snippet: Anti-human ICOS APC (Clone REA192) , Miltenyi , Cat# 130-101-723.

Techniques: Ubiquitin Proteomics, Mouse Assay, Software