Journal: Journal of applied toxicology : JAT

Article Title: Arsenic inhibits mast cell degranulation via suppression of early tyrosine phosphorylation events

doi: 10.1002/jat.3300

Figure Lengend Snippet: Arsenic inhibits RBL-2H3 cell degranulation following 1 hour exposure to DNP-BSA antigen (Ag). Effects of various concentrations of As on 1 hour, 0.00016 μg mL-1Ag-mediated degranulation were assessed as described (Hutchinson et al., 2011). In the absence of As, this antigen dose elicited an average absolute degranulation response of 12 ± 2% (SEM; ∼23% of the maximal response). Values represent means ± SEM for five independent experiments, where three replicates per dose were performed for each experiment. One-way ANOVA with Tukey's post-tests (compared to the 1 ppb sample) were performed using Graphpad Prism software; *p< 0.05, ***p< 0.001. This figure is reprinted from (Hutchinson et al., 2011) (Copyright 2010 John Wiley & Sons, Ltd.)

Article Snippet: For this assay, RBL-2H3 (1.75 × 10 6 cells per mL) were plated on 10 cm 2 tissue-culture treated, sterile dishes (Cellstar, Greiner Bio-One, Monroe, NC, USA) at a volume of 6.5 mL per well (final cell density of 1.14×10 7 cells per dish), and were incubated overnight (12-16 h) at 5% CO 2 and 37°C.

Techniques: Software

Journal: Journal of applied toxicology : JAT

Article Title: Arsenic inhibits mast cell degranulation via suppression of early tyrosine phosphorylation events

doi: 10.1002/jat.3300

Figure Lengend Snippet: F-actin ruffling during antigen-stimulated degranulation of RBL-2H3 cells. F-actin was visualized using Alexa Fluor 488 conjugated phalloidin. RBL-2H3 cells were exposed to (A) no Ag; (B) no Ag, plus 750 ppb As; (C) 0.00016 μg mL-1 Ag; (D) 0.00016 μg mL-1 Ag, plus 750 ppb As for one hour before being fixed. A representative set of images is shown.

Article Snippet: For this assay, RBL-2H3 (1.75 × 10 6 cells per mL) were plated on 10 cm 2 tissue-culture treated, sterile dishes (Cellstar, Greiner Bio-One, Monroe, NC, USA) at a volume of 6.5 mL per well (final cell density of 1.14×10 7 cells per dish), and were incubated overnight (12-16 h) at 5% CO 2 and 37°C.

Techniques:

Journal: Journal of applied toxicology : JAT

Article Title: Arsenic inhibits mast cell degranulation via suppression of early tyrosine phosphorylation events

doi: 10.1002/jat.3300

Figure Lengend Snippet: Arsenic does not inhibit A23187 Ca2+ ionophore-stimulated degranulation in RBL-2H3 cells. RBL-2H3 cells were harvested and plated as described in the “Methods” section. Cells were stimulated to degranulate for 1 h with either (A) 1.5 × 10-7 M (which caused an average absolute degranulation response of 11.5% ± 2.8% [SEM]) or (B) 2.0 × 10-7 M (which caused an average absolute degranulation response of 18.1% ± 3.2% [SEM]) A23187 Ca2+ ionophore. Resultant β-hexosaminidase was measured as described in “Methods.” Values are means ± SEM from seven independent experiments per A23187 dose, each with three replicates per experiment. No statistical significance was determined by one-way ANOVA followed by Tukey's post- test, where multiple comparisons were made to the 100 ppb sample.

Article Snippet: For this assay, RBL-2H3 (1.75 × 10 6 cells per mL) were plated on 10 cm 2 tissue-culture treated, sterile dishes (Cellstar, Greiner Bio-One, Monroe, NC, USA) at a volume of 6.5 mL per well (final cell density of 1.14×10 7 cells per dish), and were incubated overnight (12-16 h) at 5% CO 2 and 37°C.

Techniques:

Journal: Journal of applied toxicology : JAT

Article Title: Arsenic inhibits mast cell degranulation via suppression of early tyrosine phosphorylation events

doi: 10.1002/jat.3300

Figure Lengend Snippet: Arsenic does not inhibit thapsigargin (Tg) -induced degranulation. (A) Relative degranulation response of RBL-2H3 cells treated for 1hr with 4.6 nM Tg (which caused an average absolute degranulation response of 11.9 + 3.9% [SEM]), ± As. Values represent mean ± SEM of 3-5 experiments of triplicate samples per dose per experiment. A spontaneous release measurement (no Tg or As present) is depicted for reference. No significant difference was determined by one-way ANOVA. In (B) trypan blue exclusion assays, the percentage of living cells present in the 0 nM Tg control is plotted against concentration of Tg (1.5 and 4.6 nM); all samples contain 750 ppb As. Values are means of two experiments, each with triplicate samples, where data were normalized to the 0 control (with no Tg and no As, but containing Tg vehicle of 0.001% DMSO). No significant difference was determined by one-way ANOVA. (C) Short-term cytotoxicity was determined using an LDH cytotoxicity detection kit; concentration of Tg is plotted against the percentage LDH released in the absence or presence of 750 ppb As; values represent individual wells (n = 6-9), and error bars are SD. No significant difference was determined by one-way ANOVA.

Article Snippet: For this assay, RBL-2H3 (1.75 × 10 6 cells per mL) were plated on 10 cm 2 tissue-culture treated, sterile dishes (Cellstar, Greiner Bio-One, Monroe, NC, USA) at a volume of 6.5 mL per well (final cell density of 1.14×10 7 cells per dish), and were incubated overnight (12-16 h) at 5% CO 2 and 37°C.

Techniques: Concentration Assay

Journal: Journal of applied toxicology : JAT

Article Title: Arsenic inhibits mast cell degranulation via suppression of early tyrosine phosphorylation events

doi: 10.1002/jat.3300

Figure Lengend Snippet: Arsenic does not affect c48/80-induced degranulation of RBL-2H3 cells. (A) Effect of As on c48/80-mediated degranulation. Cells were pre-treated with quercetin and then co-exposed to 25 μg mL-1 c48/80 (which caused an average absolute degranulation response of 7.7% ± 0.6% [SEM]) and varying concentrations of As (0-750 ppb) for 15 minutes, followed by β-hexosaminidase quantification as described in “Methods.” Data were normalized to the 0 ppb As response value of each experiment (n = 5). (B) LDH cytotoxicity assay indicating non-cytotoxicity of 25 μg mL-1 c48/80 treatment (n = 3). Cells were treated as in (A), and LDH release was measured as described in “Methods.” In (A-B), values represent means ± SEM; statistical significance was determined by one-way ANOVA followed by Tukey's post test (in A, comparison made to 100 ppb; in B, direct comparison is made to 0 μg mL-1 c48/80); *p< 0.05, ***p< 0.001.

Article Snippet: For this assay, RBL-2H3 (1.75 × 10 6 cells per mL) were plated on 10 cm 2 tissue-culture treated, sterile dishes (Cellstar, Greiner Bio-One, Monroe, NC, USA) at a volume of 6.5 mL per well (final cell density of 1.14×10 7 cells per dish), and were incubated overnight (12-16 h) at 5% CO 2 and 37°C.

Techniques: LDH Cytotoxicity Assay

Journal: Journal of applied toxicology : JAT

Article Title: Arsenic inhibits mast cell degranulation via suppression of early tyrosine phosphorylation events

doi: 10.1002/jat.3300

Figure Lengend Snippet: Arsenic dampens Ca2+ influx into antigen-activated RBL-2H3 cells. Fluo-4/AM was used to measure Ca2+ levels within RBL-2H3 cells which had been sensitized with anti-DNP IgE (0.1μg mL-1) for 1 hr before exposure to ± 0.0002 μg mL-1 DNP-BSA Ag ± 750ppb As, at 30 minutes (A) or 1 hour (B) post-Ag stimulation. Control samples received no As or Ag. Values represent mean (normalized to 0 ppb As) ± SEM of four experiments, with 8-16 replicates per dose per experiment. Statistical significance, as compared to the 0 ppb As sample, was determined by one-way ANOVA followed by Tukey's post test; ***p<0.001

Article Snippet: For this assay, RBL-2H3 (1.75 × 10 6 cells per mL) were plated on 10 cm 2 tissue-culture treated, sterile dishes (Cellstar, Greiner Bio-One, Monroe, NC, USA) at a volume of 6.5 mL per well (final cell density of 1.14×10 7 cells per dish), and were incubated overnight (12-16 h) at 5% CO 2 and 37°C.

Techniques:

Journal: Journal of applied toxicology : JAT

Article Title: Arsenic inhibits mast cell degranulation via suppression of early tyrosine phosphorylation events

doi: 10.1002/jat.3300

Figure Lengend Snippet: Arsenic inhibits phosphorylation of phosphoinositide 3-kinase (PI3K) in RBL-2H3 cells. RBL-2H3 cells were sensitized with anti-DNP IgE (0.1μg mL-1) for 1 hr before being treated with DNP-BSA Ag (1μg mL-1) ± 750ppb As for 5 min. Levels of (A) phosphorylation of the p85 subunit of PI3K and (B) total p85 subunit of PI3K were measured with a Fast Activated Cell-based ELISA kit. Data were plotted after correction for cell number (by use of crystal violet staining). Values represent mean (normalized to 0 ppb As) ± SEM of 3-4 experiments of triplicate samples. Statistical significance was determined by one sample t-test; **p<0.01.

Article Snippet: For this assay, RBL-2H3 (1.75 × 10 6 cells per mL) were plated on 10 cm 2 tissue-culture treated, sterile dishes (Cellstar, Greiner Bio-One, Monroe, NC, USA) at a volume of 6.5 mL per well (final cell density of 1.14×10 7 cells per dish), and were incubated overnight (12-16 h) at 5% CO 2 and 37°C.

Techniques: In-Cell ELISA, Staining

Journal: Journal of applied toxicology : JAT

Article Title: Arsenic inhibits mast cell degranulation via suppression of early tyrosine phosphorylation events

doi: 10.1002/jat.3300

Figure Lengend Snippet: Arsenic inhibits phosphorylation of Syk kinase. RBL-2H3 cells were sensitized with anti-DNP IgE (0.1μg mL-1) for 1 hr before being treated with DNP-BSA Ag (1μg mL-1) ± 750 ppb As for 5 min. Phosphorylation of Syk was measured with a PathScan ® Phospho-Syk (panTyr) Sandwich ELISA Kit. Values are expressed as % increase over spontaneous (no Ag) control samples and represent mean ± SEM of four experiments, each of triplicate samples. Statistical significance was determined by one sample t-test; *p<0.05

Article Snippet: For this assay, RBL-2H3 (1.75 × 10 6 cells per mL) were plated on 10 cm 2 tissue-culture treated, sterile dishes (Cellstar, Greiner Bio-One, Monroe, NC, USA) at a volume of 6.5 mL per well (final cell density of 1.14×10 7 cells per dish), and were incubated overnight (12-16 h) at 5% CO 2 and 37°C.

Techniques: Sandwich ELISA