Journal: Journal of Medical Entomology

Article Title: Mouse chymase mast cell protease-4 facilitates blood feeding of Aedes aegypti (Diptera: Culicidae) mosquitoes

doi: 10.1093/jme/tjaf137

Figure Lengend Snippet: Sensitized serum enhances mast cell degranulation and chymase production, and chyamse mMCP-4 degrades some Aedes aegypti salivary proteins (SGPs). A) β-hexosaminidase or B) chymase enzyme activity was measured in BMMCs incubated with serum from mosquito-sensitized or naïve mice and stimulated with SGPs at the indicated concentrations (0, 1, 10, or 100 ng/ml). Lipopolysaccharide (1 μg/ml) was used as a positive control. “+” or “−” indicates the presence or absence of non-sensitized serum, sensitized serum, and SGPs for each condition. Data are presented as means ± SEM from 2 independent experiments with 3 technical replicates each ( n = 6 per condition). C) 20 μg of the extracted SGPs were incubated with 0.01 μg of recombinant mouse tryptase MCP-6 or chymase MCP-4 for overnight (approximately 18 h). SDS-page gels were stained with coomassie blue. Proteins marked with arrow were identified by liquid chromatograph mass spectrometer. Statistical analysis was performed using ordinary 1-way ANOVA followed by Sidak’s multiple comparisons test for preselected pairs. * P < 0.05; ** P < 0.01; **** P < 0.0001; ns, not significant.

Article Snippet: To evaluate the enzymatic degradation of mosquito SGPs by mouse mast cell proteases, 20 μg of SGPs were incubated with either 0.01 μg of recombinant mouse tryptase MCP-6 (Cat# 3736-SE, R&D Systems) or 0.01 μg of recombinant mouse chymase MCP-4 Reactions were carried out at room temperature (∼22 °C) for approximately 18 h to allow for proteolytic cleavage.

Techniques: Activity Assay, Incubation, Positive Control, Recombinant, SDS Page, Staining, Mass Spectrometry

Journal: Immunity

Article Title: Genetic tracing reveals transcription factor Foxp3-dependent and Foxp3-independent functionality of peripherally induced Treg cells.

doi: 10.1016/j.immuni.2022.05.010

Figure Lengend Snippet: Figure 5. Colonic Treg cells have Foxp3-dependent and Foxp3-independent suppressive functions (A) Experimental design. Female mice of indicated genotypes were treated with DT (1mg via intraperitoneal injection on d0, d1, and d7) and analyzed 2 weeks after the start of treatment. (B and C) Intracellular cytokine staining following 3-h ex vivo re-stimulation. Pooled data from four experiments with n = 8–25 mice per group. (D) H&E staining of colonic sections from indicated genotypes and colitis scores (0–3). Pooled data from two independent experiments with n = 5–11 mice per group. (E) Flow cytometry of mast cells in the colonic lamina propria (LP) and intraepithelial (IEL) fractions. Pooled data from 4 (LP) or 3 (IEL) independent experiments with n = 8–25 mice per group (LP) or n = 7–15 mice per group (IEL). (F) Representative whole-mount immunofluorescence (IF) images of the proximal colon of mice of the indicated genotypes stained with anti-Mcpt1 (red) and anti- EpCam (blue). Scale bars, 50mm. Scale bar of inset below, 10mm. (G) Total CD4 and CD8 T cell counts in the spleen and colonic LP analyzed by flow cytometry. Pooled data from 8 experiments with n = 16–31 mice per group. p values from Brown-Forsythe and Welch ANOVA(*p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001). All error bars denote the mean (SEM). See also Figure S5.

Article Snippet: Antibodies for intestinal mast cell immunofluorescence The following antibodies were used for intestinal mast cell visualization: Polyclonal rabbit anti-mouse GFP Alexa Fluor 488 (ThermoFisher Cat #A-21311), rat anti-mouse Mcpt1 (R&D systems: MAB5146) and rat anti-mouse EpCAM Alexa Fluor 647 (BioLegend: Cat #118212).

Techniques: Injection, Staining, Ex Vivo, Flow Cytometry, Cytometry