dapi exclusion  (Sony Biotechnology)

Journal: Frontiers in Immunology

Article Title: Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro

doi: 10.3389/fimmu.2021.737988

Figure Lengend Snippet: SHIP1 activity is necessary for Siglec-8–induced eosinophil cell death. (A) Eosinophils were treated with anti-Siglec-8 (clone 2C4) or isotype control mAb (IgG1) for the indicated durations in the absence of IL-5 (No IL-5) or after being primed in the presence of 30 ng/ml IL-5 for 18–24 hr. The cells were then lysed, and the lysates separated by SDS-PAGE and transferred to a PVDF membrane. Tyrosine-phosphorylated proteins were detected using a phosphotyrosine-specific antibody. Blots are representative of three independent experiments. (B) IL-5–primed eosinophils were pretreated with pharmacological inhibitors of SHP-1/2 (NSC-87877, GS-493, BVT-948), SHIP1 (3-AC), or SHIP2 and SHIP1 (AS1938909) at the indicated concentrations for 30 min prior to treatment with anti-Siglec-8 (open circles) or isotype control mAb (filled circles) for 18–24 hr. Cell viability was then assessed by annexin V and DAPI staining by flow cytometry and normalized to the cell viability measured in untreated samples. Dotted lines indicate viability levels with no pharmacological inhibition. Data represent means ± standard deviations of 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to within–antibody stimulation group vehicle control sample. # p < 0.05, #### p < 0.0001 relative to isotype control sample at the same pharmacological inhibitor concentration.

Article Snippet: Purity and viability were consistently greater than 95% as determined by Siglec-8 staining and DAPI (ThermoFisher Scientific, Waltham, MA) exclusion ( ).

Techniques: Activity Assay, Control, SDS Page, Membrane, Staining, Flow Cytometry, Inhibition, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro

doi: 10.3389/fimmu.2021.737988

Figure Lengend Snippet: SFKs and Syk are necessary components of the Siglec-8 signaling cascade. (A) Eosinophils were primed with IL-5 and pretreated with pharmacological inhibitors that act on SFKs (PP1, SU6656) at the indicated concentrations for 30 min prior to treatment with anti-Siglec-8 (open circles) or isotype control mAb (filled circles) for 18–24 hr. Cell viability was then assessed by annexin V and DAPI staining by flow cytometry and normalized to that of untreated samples. Dotted lines indicate viability levels with no pharmacological inhibition. Data represent means ± standard deviations of 3 and 5 independent experiments, respectively. (B) Primed eosinophils were incubated with anti-Siglec-8 (2C4) or isotype control mAb (IgG1) for the indicated durations (in minutes) prior to lysis and the subsequent separation and detection of phospho-SFK (pY419) and total Src. Upper panel: representative western blot. Lower panel: quantified band intensities representing the means ± standard deviations of 4 independent experiments. Band intensities were normalized to those of untreated control samples. **p < 0.01, ****p < 0.0001 vs. isotype control samples at the same time point. (C) Primed eosinophils were pretreated with the Syk inhibitors R406 or OXSI-2 at the indicated concentrations for 30 min prior to treatment with anti-Siglec-8 (open circles) or isotype control mAb (filled circles) for 18–24 hr. Cell viability was assessed as in (A) . Data represent means ± standard deviations of 3 independent experiments. (A, C) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to within–antibody stimulation group vehicle control sample. ## p < 0.01, ### p < 0.001, #### p < 0.0001 relative to isotype control sample at the same pharmacological inhibitor concentration.

Article Snippet: Purity and viability were consistently greater than 95% as determined by Siglec-8 staining and DAPI (ThermoFisher Scientific, Waltham, MA) exclusion ( ).

Techniques: Control, Staining, Flow Cytometry, Inhibition, Incubation, Lysis, Western Blot, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro

doi: 10.3389/fimmu.2021.737988

Figure Lengend Snippet: Btk is activated late in the Siglec-8 signaling pathway and is necessary for Siglec-8–induced eosinophil cell death. Human eosinophils were stimulated with IL-5 and pretreated with the indicated concentrations of the Btk inhibitors ibrutinib (A) or acalabrutinib (B) for 30 min prior to treatment with anti-Siglec-8 (open circles) or isotype control (filled circles) for 18–24 h. Eosinophil viability was then assessed by annexin V/DAPI staining as before. Dotted lines indicate viability levels with no pharmacological inhibition. Plots represent mean normalized eosinophil viability ± standard deviations for 3 independent experiments. **p < 0.01, **** p < 0.0001 relative to within–antibody stimulation group vehicle control sample. # p < 0.05, #### p < 0.0001 relative to isotype control sample at the same pharmacological inhibitor concentration. (C) Eosinophils were treated with anti-Siglec-8 mAb (2C4, white columns), isotype control mAb (IgG1, black columns), or left untreated for the indicated durations before lysis and separation of proteins by SDS-PAGE. Immunoblotting for phospho-Btk, total Btk, and β-actin was performed. The blot is representative of 3 independent experiments. Quantitation of phospho-Btk relative to β-actin or total Btk normalized to the untreated eosinophils is shown. The data represent means ± standard deviations for 3 independent experiments. *p < 0.05; **p < 0.01.

Article Snippet: Purity and viability were consistently greater than 95% as determined by Siglec-8 staining and DAPI (ThermoFisher Scientific, Waltham, MA) exclusion ( ).

Techniques: Control, Staining, Inhibition, Concentration Assay, Lysis, SDS Page, Western Blot, Quantitation Assay

Journal: Frontiers in Immunology

Article Title: Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro

doi: 10.3389/fimmu.2021.737988

Figure Lengend Snippet: The activities of MEK1, ERK1/2, and PAK1 are necessary for Siglec-8–induced eosinophil cell death. Peripheral blood eosinophils were stimulated with 30 ng/ml IL-5 overnight and pretreated with the indicated concentration of the MEK1/2 inhibitors U0126 (A) or PD98059 (B) , ERK1/2 inhibitor SCH772984 (C) , c-Raf inhibitor GW5074 (D) , or PAK1/2 inhibitor AZ13705339 (E) for 30 min prior to 18–24-h treatment with anti-Siglec-8 (open circles) or isotype control mAb (filled circles). Cell viability was then assessed by annexin V/DAPI staining by flow cytometry. Dotted lines indicate viability levels with no pharmacological inhibition for inhibitors plotted on a logarithmic scale. Plots represent the mean percentage of viable eosinophils normalized to the viability of untreated eosinophils ± standard deviations for 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to within–antibody stimulation group vehicle control sample. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 relative to isotype control sample at the same pharmacological inhibitor concentration. (F) IL-5–primed eosinophils were treated with anti-Siglec-8 mAb or isotype control mAb (mIgG1) for the indicated durations prior to lysis and detection of phospho- (pT202/pY204) or total ERK1/2 using an automated protein separation and immunodetection platform. Blot is representative and quantified data represent the means and standard deviations of three independent experiments. **p < 0.01; ****p < 0.0001 vs. isotype control at the same time point.

Article Snippet: Purity and viability were consistently greater than 95% as determined by Siglec-8 staining and DAPI (ThermoFisher Scientific, Waltham, MA) exclusion ( ).

Techniques: Concentration Assay, Control, Staining, Flow Cytometry, Inhibition, Lysis, Immunodetection

Journal: Frontiers in Immunology

Article Title: Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro

doi: 10.3389/fimmu.2021.737988

Figure Lengend Snippet: The PLC-PKC signaling axis and sphingomyelin metabolism are necessary for Siglec-8–induced eosinophil cell death. Peripheral blood eosinophils were stimulated with 30 ng/ml IL-5 overnight and pretreated with the indicated concentration of the PKC inhibitor GF109203x (A) , the PLC inhibitor U73122 (circle symbols) or pharmacologically inactive analog U73343 (square symbols) (B) , or the functional acid sphingomyelinase and acid ceramidase inhibitor desipramine (C) for 30 min prior to 18–24-h treatment with anti-Siglec-8 (open symbols) or isotype control mAb (filled symbols). Cell viability was then assessed by annexin V/DAPI staining by flow cytometry. Dotted lines indicate viability levels with no pharmacological inhibition for inhibitors plotted on a logarithmic scale. Plots represent the mean percentage of viable eosinophils normalized to the viability of untreated eosinophils ± standard deviations for 3 independent experiments. *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001 relative to within–antibody stimulation group vehicle control sample. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 relative to isotype control sample at the same pharmacological inhibitor concentration.

Article Snippet: Purity and viability were consistently greater than 95% as determined by Siglec-8 staining and DAPI (ThermoFisher Scientific, Waltham, MA) exclusion ( ).

Techniques: Concentration Assay, Functional Assay, Control, Staining, Flow Cytometry, Inhibition

Journal: Frontiers in Immunology

Article Title: Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro

doi: 10.3389/fimmu.2021.737988

Figure Lengend Snippet: Actin depolymerization is necessary and sufficient for CD11b surface upregulation and activation, and Btk is involved in CD11b activation but not upregulation. (A) Eosinophils were pretreated with the indicated disruptors of cytoskeletal dynamics for 30 min prior to treatment with anti-Siglec-8 or isotype control mAb. After 18–24 h, eosinophil cell viability was assessed by annexin V/DAPI staining as before, and normalized to the isotype control sample within each treatment. Individual results, means, and standard deviations are shown for 4 independent experiments. ***p < 0.0001 relative to vehicle control–pretreated eosinophils. (B) Following pretreatment with the indicated agents for 30 min and treatment with anti-Siglec-8 (filled histograms) or isotype control mAb (empty histograms) for 120 min, surface expression of CD11b was assessed by flow cytometry on live (DAPI-negative) eosinophils. Isotype control mAb-stained samples are included for reference. Histograms are representative of 3 independent experiments. (C) CD11b expression levels on eosinophils treated with anti-Siglec-8 (white columns) or isotype control mAb (black columns) were quantified and normalized to those of control eosinophils. Data represent means ± standard deviations of 3 independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to within–antibody stimulation group vehicle control sample. ### p < 0.001, #### p < 0.0001 relative to within–pretreatment group isotype control sample. (D) Following pretreatment with the indicated agents for 30 min and treatment with anti-Siglec-8 (white columns) or isotype control mAb (black columns) for 120 min, conformational activation of CD11b on live (DAPI-negative) eosinophils was measured by flow cytometry. Activated CD11b levels were normalized to those found on control eosinophils. Data represent means ± standard deviations of 4 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 relative to within–antibody stimulation group vehicle control sample. # p < 0.05, ## p < 0.01, ### p < 0.001 relative to within–pretreatment group isotype control sample.

Article Snippet: Purity and viability were consistently greater than 95% as determined by Siglec-8 staining and DAPI (ThermoFisher Scientific, Waltham, MA) exclusion ( ).

Techniques: Activation Assay, Control, Staining, Expressing, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro

doi: 10.3389/fimmu.2021.737988

Figure Lengend Snippet: In addition to molecules involved in CD11b surface upregulation and activation, actin polymerization is necessary for Siglec-8–induced ROS production. Following pretreatment with the indicated agents for 30 min and treatment with anti-Siglec-8 (filled histograms) or isotype control mAb (empty histograms) for 120 min, ROS production was assessed by flow cytometry on live (DAPI-negative) eosinophils using DHR 123. Controls include samples lacking the DHR 123 probe, or those with the stain but maintained at 4°C or 37°C without stimulation (No stim). DHR 123 fluorescence was normalized to that of anti-Siglec-8 mAb–stimulated eosinophils that were not treated with any pharmacological inhibitor. Data represent means ± standard deviations of 3 independent experiments. ****p < 0.0001 relative to within–antibody stimulation group vehicle control sample. ## p < 0.01, #### p < 0.0001 relative to within–pretreatment group isotype control sample.

Article Snippet: Purity and viability were consistently greater than 95% as determined by Siglec-8 staining and DAPI (ThermoFisher Scientific, Waltham, MA) exclusion ( ).

Techniques: Activation Assay, Control, Flow Cytometry, Staining, Fluorescence