apc anti-mouse il-13 85brd  (Thermo Fisher)

Journal: Nature Communications

Article Title: Orai inhibition modulates pulmonary ILC2 metabolism and alleviates airway hyperreactivity in murine and humanized models

doi: 10.1038/s41467-023-41065-4

Figure Lengend Snippet: A Block of endogenous SOCE in FACS-sorted pulmonary ILC2s after exposure to compound 5D measured by flow cytometry. Cells were pretreated with thapsigargin to deplete the intracellular Ca 2+ stores before addition of Ca 2+ and 5D exposure. B FACS-sorted pulmonary murine ILC2s were cultured for 24 h with survival cytokines rmIL-2, rmIL-7 and with rmIL-33 or PBS control for 24 h. 5D or the vehicle control were then added to the wells for an additional 48 h. C After 48 h, cells were collected and Ki67 as a measure of proliferation was assessed and quantified by flow cytometry presented as mean ± SEM. n = 3 biologically independent samples. D Supernatant was collected and secreted cytokines were measured by Legendplex (S2A). Presented as mean ± SEM. n = 3 biologically independent samples. E Representative flow cytometry plots of IL-5 + and IL-13 + ILC2s from ILC2s cultured with 5D or the vehicle and corresponding quantitation presented as mean ± SEM. n = 3 biologically independent samples. F Pulmonary ILC2s were sorted from wild-type (WT), Orai1 −/− and Orai1/2 −/− mice and cultured with and without tamoxifen for 48 h. Cells were collected, counted, and cultured for an additional 48 h. Supernatant was collected and proinflammatory cytokines were measured by Legendplex (S2B). Presented as mean ± SEM. n = 3 biologically independent samples. G ILC2s were collected, and intracellular IL-5 and IL-13 production was measured and quantified by flow cytometry (S2C). Presented as mean ± SEM. n = 3 biologically independent samples. Data are representative of 2 independent experiments and are presented as means ± SEM. Source data are provided as a Source data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. ILC2 cell image designed with Servier Medical Art.

Article Snippet: APC anti-mouse IL-13 (85BRD, Thermofisher), PE anti-mouse IL-5 (TRFK5, BioLegend) were used.

Techniques: Blocking Assay, Flow Cytometry, Cell Culture, Quantitation Assay, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Orai inhibition modulates pulmonary ILC2 metabolism and alleviates airway hyperreactivity in murine and humanized models

doi: 10.1038/s41467-023-41065-4

Figure Lengend Snippet: A Schematic of the mitochondrial electron transport chain (mtETC) and the associated complexes. B Heatmap of statistically significant differentially expressed genes associated with the mtETC in ILC2s cultured with 5D or the vehicle. Arrow colors coordinate with the associated complex in ( A ). C Flow cytometry histogram and quantification of mitochondrial mass measured by Mitotracker Green in ILC2s cultured with 5D or the vehicle presented as mean ± SEM. n = 3 biologically independent samples. D Flow cytometry histogram and quantification of mitochondrial membrane potential measured by Mitotracker Red in ILC2s cultured with 5D or the vehicle presented as mean ± SEM. n = 3 biologically independent samples. E Total cellular reactive oxygen species (ROS) in treated and untreated ILC2s measured by microplate fluorescence presented as mean ± SEM. n = 3 biologically independent samples. F Flow cytometry histogram and quantification of mitochondrial ROS in ILC2s cultured with 5D or the vehicle as measured by Mitosox presented as mean ± SEM. n = 3 biologically independent samples. G NADH/NAD+ ratio quantification in ILC2s cultured with 5D or the vehicle measured by microplate reader presented as mean ± SEM. n = 3 biologically independent samples. H ATP production presented as OCR measured by Seahorse Mitostress Test assay presented as mean ± SEM. n = 3 biologically independent samples. I Representative flow cytometry plots and quantification of intracellular IL-5 production in ILC2s cultured with the vehicle, 5D, or 5D and the addition of antioxidant NAC presented as mean ± SEM. n = 3 biologically independent samples. J Representative flow cytometry plots and quantification of intracellular IL-13 production in ILC2s cultured with the vehicle, 5D, or 5D and the addition of antioxidant NAC presented as mean ± SEM. n = 3 biologically independent samples. K Representative flow cytometry plots and quantification of proliferation in ILC2s cultured with the vehicle, 5D, or 5D and the addition of antioxidant NAC presented as mean ± SEM. n = 3 biologically independent samples. Data are representative of two independent experiments and are presented as means ± SEM. Source data are provided as a Source data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. Figure 5A was created with BioRender.com.

Article Snippet: APC anti-mouse IL-13 (85BRD, Thermofisher), PE anti-mouse IL-5 (TRFK5, BioLegend) were used.

Techniques: Cell Culture, Flow Cytometry, Membrane, Fluorescence, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Orai inhibition modulates pulmonary ILC2 metabolism and alleviates airway hyperreactivity in murine and humanized models

doi: 10.1038/s41467-023-41065-4

Figure Lengend Snippet: A FACS-sorted human blood ILC2s were cultured with rhIL-2, rhIL-7, and rhIL-33 for 24 h. 5D or vehicle were added to the culture for an additional 48 h. B Flow cytometry histogram and quantification of Orai1 expression after 24 h with survival cytokines in ( A ) presented as mean ± SEM. n = 4 biologically independent samples. C Flow cytometry histogram and quantification of Orai2 expression after 24 h with survival cytokines in ( A ) presented as mean ± SEM. n = 4 biologically independent samples. D After 48 h with 5D or vehicle, supernatant was collected and IL-5 and IL-13 levels were measured by ELISA. n = 6 biologically independent samples. E FACS-sorted human blood ILC2s from healthy donors were adoptively transferred into Rag2 −/− γc −/− mice. Mice were intranasally challenged with rhIL-33 (1 μg) or PBS and treated with i.p. injections of 5D or vehicle days 0, 1, and 2. Mice were euthanized on day 3 and AHR ( F ) was measured presented as mean ± SEM. n = 4 biologically independent samples. G Total number of eosinophils infiltrating the BAL presented as mean ± SEM. n = 4 biologically independent samples. H Total number of human ILC2s found in the lung presented as mean ± SEM. n = 4 biologically independent samples. Data are representative of two independent experiments and are presented as means ± SEM. Source data are provided as a Source data file. A two-tailed Student’s t test for unpaired data was applied for comparisons between two groups, except for multi-group comparisons where Tukey’s multiple comparison one-way ANOVA tests were used. Mouse and human ILC2 cell images designed with Servier Medical Art.

Article Snippet: APC anti-mouse IL-13 (85BRD, Thermofisher), PE anti-mouse IL-5 (TRFK5, BioLegend) were used.

Techniques: Cell Culture, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: Developmental cell

Article Title: A comprehensive roadmap of murine spermatogenesis defined by single-cell RNA-seq

doi: 10.1016/j.devcel.2018.07.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: APC anti-mouse IL-13 Antibody , Novus , Cat#011818.

Techniques: Recombinant, Reverse Transcription, SYBR Green Assay, Gene Expression, Software