Journal: PLoS ONE

Article Title: Cellular Size as a Means of Tracking mTOR Activity and Cell Fate of CD4+ T Cells upon Antigen Recognition

doi: 10.1371/journal.pone.0121710

Figure Lengend Snippet: a) Relative expression of IFN-gamma, IL-17a and IL-17f mRNA transcripts were determined from mTOR hi and mTOR lo populations immediately after sorting stimulated 5c.c7 Rag –/— splenocytes. Error bars show ± S.D. from 3 independent experiments, and mTOR hi values were set to 1. b-d ) Sorted populations of cells were cultured in media supplemented with IL-2 for 3 days prior to assessment. b ) Expression of chemokine receptor, CXCR3, and LFA-1 subunit CD11a was determined by flow cytometry. MFI per population is shown at the top of each FACs plot. c ) Relative expression of Foxp3 mRNA transcript ± S.D. was determined from the mTOR lo population compared to the mTOR hi cells. The graph was generated from 3 independent experiments, and mTOR hi values were set to 1. d) Foxp3 protein expression was determined from sorted mTOR lo and mTOR hi populations 3 days after culture in IL-2 alone or IL-2 + TGF-ß. Bar graphs to the right depict % Foxp3+ cells ± S.D. generated in each population from 3 independent experiments. e ) mTOR lo Foxp3+ regulatory T cells possess the ability to suppress the proliferation of naïve CD4+ T cells. Sorted mTOR hi or mTOR lo populations were cultured in media supplemented with IL-2 for 3 days prior to use in the suppression assay. The percentage of non-divided responders was determined by CFSE dilution 72hrs after culture of 1:2 suppressors: responders in direct contact or in transwell suppression assays. The graphs to the right depict the percentage of non-divided responders after culture with mTOR hi or mTOR lo ‘suppressors’ from 3 independent experiments. Connecting lines show values from sorted populations from the same experiment. The data are representative of at least 3 independent experiments or show cumulative results.

Article Snippet: Anti-Bcl-2 (BCL/10C4) and anti-CXCR3 (CXCR3-173) were purchased from Biolegend.

Techniques: Expressing, Cell Culture, Flow Cytometry, Generated, Suppression Assay

Journal: Allergy

Article Title: Butyrate as a bioactive human milk protective component against food allergy

doi: 10.1111/all.14625

Figure Lengend Snippet: Evaluation of direct immunoregulatory action of HM butyrate on human peripheral blood mononuclear cells (PBMCs): modulation of mono‐dendritic cell expression. PBMCs from children with IgE‐mediated FA were stimulated with butyrate HM median concentration (0.75 mM) or with specific allergen (BLG, OVA, PN) (100 μL/well of each allergen) in the presence or in the absence of butyrate for 48 h (A‐B). CD45hi CD14+ monocytes were stained with flow cytometric markers CD206+ or CD86+ to discern the CD206 M2 type from CD86 M1 type of total monocytes. A representative dot plot panel (left panel) is presented. The graph on the right reports the mean ± SD of ratios of M1 vs M2 peripheral monocytes (CD86/CD206 ratio) from different FA patients (ANOVA; *** P < .001 vs CTRL) (B). Purified CD14+ monocytes from IgE‐mediated FA were cultured with regular medium containing GM‐CSF (50 ng/mL) and IL‐4 (1000 U/mL), with or without butyrate HM median concentration (0.75 mM). After 7 d, cells were collected, stained with the reported antibodies, and analyzed by flow cytometry (C‐E). Representative example of dot plots of iDCs generated in the presence or in the absence of butyrate (C). The bars graph reports mean and standard deviation of % of CD14‐CD1a+ from independent experiments using different IgE‐mediated FA (ANOVA; *** P < .001 vs CTRL). (D). Immature DCs were collected and exposed to LPS‐activating stimulus (1 μg/mL). After 24 h, cells were stained for CD86 maturation marker. Bar graphs report the percentage of positive cells for the indicated markers in gated viable CD14‐CD1a+ cells (ANOVA; *** P < .001 vs CTRL) (E). Phenotypical characterization of iDCs generated in the presence or in the absence of 0.75 mM of butyrate for 7 d in healthy subjects (left panel) and in IgE‐mediated FA (right panel). Bar graph reports mean and standard deviation of % of CD11b+ CD103+ from independent experiments using IgE‐mediated FA (ANOVA; *** P < .001 vs CTRL). (F‐G). Butyrate‐DC had no effect on regulatory cell differentiation. Naïve CD4+ T cells from IgE‐mediated FA were cultured with immature or mature DCs generated in the presence of butyrate at 1:10 DC:T ratio. After being cultured for 5 d, cells were collected. CD4+CD25+CD127 dim/ⁿeg Tregs were identified and enumerated with a flow cytometer (H). The bar graphs report the percentage of positive cells for the indicated markers

Article Snippet: Single‐cell suspensions from PBMCs were stained with mAb against human CD14 (HCD14; BioLegend), human CD206 (DCN228; Miltenyi Biotec), human/mouse CD11b (M1/70.15.11.5; Miltenyi Biotec), human CD1a (HI149; Miltenyi Biotec), human CD1a (HI149; Miltenyi Biotec), human CD86 (REA968; Miltenyi Biotec), human CCR7 (REA108; Miltenyi Biotec), human CD45 RA (T6D11; Miltenyi Biotec), CD4 (A161A1; BioLegend), and human CD103 (REA803; Miltenyi Biotec).

Techniques: Expressing, Concentration Assay, Staining, Purification, Cell Culture, Flow Cytometry, Generated, Standard Deviation, Marker, Cell Differentiation

Journal: Cells

Article Title: Lasting DNA Damage and Aberrant DNA Repair Gene Expression Profile Are Associated with Post-Chronic Cadmium Exposure in Human Bronchial Epithelial Cells

doi: 10.3390/cells8080842

Figure Lengend Snippet: DNA repair genes and their associated DNA repair-related pathways investigated in this study.

Article Snippet: Antibodies were purchased from Santa Cruz Biotechnology: ATR (sc-515173); ATM (sc-377293); CDK7 (sc-365075); ERCC1 (sc-17809); LIG3 (sc-390922); NEIL1 (sc-271164); NEIL3 (sc-393703); PARP1 (sc-8007); PMS2 (sc-25315); SMUG1 (sc-377370); SUMO1 (sc-5308), GeneTex: ERCC2 (GTX108948); TP53 (GTX70214), Sigma-Aldrich: ACTB (A5441), ABClonal: H2AFX (A11361), and Cell Signaling Technology: H3 (4499).

Techniques: Non-Homologous End Joining, Translesion Synthesis, Modification