Journal: Toxicology

Article Title: Evaluation of non-invasive biomonitoring of 2,4-Dichlorophenoxyacetic acid (2,4-D) in saliva

doi: 10.1016/j.tox.2018.08.003

Figure Lengend Snippet: Transwell system with serous-acinar cells grown on a Transwell insert that separates the apical (saliva) and basolateral (plasma) compartments (A). Confocal microscopy of SGECs grown on Nunc Tech II chamber slides 6 days post-seeding (B). DAPI (nuclear stain) shown in blue (top left) and Crumbs apical marker of polarity (CRB3) in green (top center). Overlay of nuclear stain and CRB3 (top right), 3-D rendering (bottom left), cross-sectional x–z axis (bottom center), and cross-sectional y–z axis (bottom right) identify CRB3 localization on the apical surface of SGECs. Scale bar = 10 µm (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Article Snippet: Anti-CRB3 rat primary monoclonal antibody was purchased from MyBioSource (Cat # MBS588104) and goat anti-rat IgG secondary antibody-Alexa Fluor 488 conjugate was purchased from Invitrogen (Cat # {"type":"entrez-nucleotide","attrs":{"text":"A11006","term_id":"492389","term_text":"A11006"}} A11006 ).

Techniques: Confocal Microscopy, Staining, Marker

Journal: Cell Insight

Article Title: Cannabinoid receptor 2 signal promotes type 2 immunity in the lung

doi: 10.1016/j.cellin.2023.100124

Figure Lengend Snippet: Genetic deletion of CB2 does not affect the normal development or immune homeostasis of the lung. (A to C) Normal lung development with the CB2 deletion. (A) Gross appearance of the lung tissues (left lobe) of adult CB2 −/− and control CB2 +/+ littermates. (B) Tissue weights (left lobe) were measured. n = 6, mean ± SEM, n.s., not significant (Student's t -test). (C) Representative images of the lung tissue sections of CB2 +/+ and CB2 −/− mice assessed by hematoxylin and eosin (H&E) staining. (D) The CB2 deletion does not affect tissue levels of the endocannabinoid 2-AG. 2-AG tissue levels in the lungs of CB2 +/+ and CB2 −/− mice were compared by the ELISA analysis. n = 6, mean ± SEM, n.s., not significant (Student's t -test). (E and F) Immune cell populations are not affected by the CB2 deletion. (E) ILC2s and other immune cell types in the lungs of CB2 +/+ and CB2 −/− mice were examined by the FACS analysis. n = 5, mean ± SEM, n.s., not significant (Student's t -test). (F) Representative FACS gating of eosinophils (CD45 + CD11b + CD11c − Siglec-F + ) and alveolar macrophages (CD45 + CD11b + CD11c + Siglec-F + ) in the lung.

Article Snippet: The tissues were blocked with PBS/0.2% Triton X-100/10% DMSO/5% normal donkey serum at room temperature overnight and then immunolabeled with rat anti-Siglec-F primary antibody (BD Biosciences, Cat#552125, RRID:AB_394340; final concentration of 1 μg/ml) in PBS/0.1% Tween 20/10 μg/ml heparin/5% normal donkey serum at room temperature for 72 h. The tissues were washed with PBS/0.1% Tween 20/10 μg/ml heparin at room temperature for 12 h, with the fresh buffer changed every 2 h. The tissues were further immunolabeled with Alexa Fluor 647-conjugated donkey anti-rat IgG secondary antibody (Thermo Fisher Scientific, Cat#A78947, RRID:AB_2910635; final concentration of 2 μg/ml) in PBS/0.1% Tween 20/10 μg/ml heparin/5% normal donkey serum at room temperature for 72 h. The tissues were washed with PBS/0.1% Tween 20/10 μg/ml heparin at room temperature for 48 h, with the fresh buffer changed every 8 h. All the incubation steps were performed with gentle rotation.

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Cell Insight

Article Title: Cannabinoid receptor 2 signal promotes type 2 immunity in the lung

doi: 10.1016/j.cellin.2023.100124

Figure Lengend Snippet: Genetic deletion of CB2 mitigates the IL-33-elicited type 2 immunity in the lung. (A and B) Wild-type mice were intranasally treated with IL-33 or control saline. (A) ILC2s were FACS-sorted from the saline-treated or IL-33-treated lung tissues, and CB2 mRNA levels in ILC2s were compared by the qPCR analysis. n = 3, mean ± SEM, n.s., not significant (Student's t -test). (B) 2-AG levels in the saline-treated or IL-33-treated lung tissues were quantified by the ELISA analysis. n ​= ​4, mean ​± ​SEM, ∗ p < 0.05 (Student's t -test). (C to K) CB2 −/− and control CB2 +/+ littermates were subjected to intranasal IL-33 treatment. (C) ILC2s in the IL-33-treated lungs of CB2 +/+ and CB2 −/− mice were examined by the FACS analysis. n = 7, mean ± SEM, n.s., not significant (Student's t -test). (D) mRNA levels of IL-4 , IL-5 , and IL-13 in the lung tissues of CB2 +/+ and CB2 −/− mice were determined by the qPCR analysis and normalized to the saline-treated CB2 +/+ condition. n ​= ​5 for saline-treated condition and n ​= ​15 for IL-33-treated condition, mean ​± ​SEM, ∗ p < 0.05 (two-way ANOVA test). (E) Protein levels of IL-5 and IL-13 in the IL-33-treated lungs of CB2 +/+ and CB2 −/− mice were quantified by the ELISA analysis. n ​= ​3, mean ​± ​SEM, ∗ p < 0.05 (Student's t -test). (F and G) IL-33-elicited immune response in the lung was visualized by advanced 3D imaging. The intact, unsectioned lungs (left lobe) of CB2 +/+ and CB2 −/− mice were processed for the whole-tissue anti-Siglec-F immunolabeling. (F) Representative 3D-projection images at 1.26 × magnification of the lightsheet imaging were shown. (G) The density of Siglec-F + immune cells was quantified. n ​= ​5, mean ​± ​SEM, ∗ p < 0.05 (two-way ANOVA test). (H and I) Eosinophil recruitment in the lungs of CB2 +/+ and CB2 −/− mice was assessed by the FACS analysis. (H) Representative FACS gating of eosinophils (CD45 + CD11b + CD11c − Siglec-F + ) in the IL-33-treated condition. (I) The percentage of eosinophils in total immune cells was quantified. n ​= ​5 for saline-treated condition and n ​= ​12 for IL-33-treated condition, mean ​± ​SEM, ∗ p < 0.05 (two-way ANOVA test). (J and K) The lung tissues of CB2 +/+ and CB2 −/− mice were assessed by hematoxylin and eosin (H&E) staining. (J) Representative images were shown. (K) Histologic scores were determined. n ​= ​5 for saline-treated condition and n ​= ​7 for IL-33-treated condition, mean ​± ​SEM, ∗ p < 0.05 (two-way ANOVA test).

Article Snippet: The tissues were blocked with PBS/0.2% Triton X-100/10% DMSO/5% normal donkey serum at room temperature overnight and then immunolabeled with rat anti-Siglec-F primary antibody (BD Biosciences, Cat#552125, RRID:AB_394340; final concentration of 1 μg/ml) in PBS/0.1% Tween 20/10 μg/ml heparin/5% normal donkey serum at room temperature for 72 h. The tissues were washed with PBS/0.1% Tween 20/10 μg/ml heparin at room temperature for 12 h, with the fresh buffer changed every 2 h. The tissues were further immunolabeled with Alexa Fluor 647-conjugated donkey anti-rat IgG secondary antibody (Thermo Fisher Scientific, Cat#A78947, RRID:AB_2910635; final concentration of 2 μg/ml) in PBS/0.1% Tween 20/10 μg/ml heparin/5% normal donkey serum at room temperature for 72 h. The tissues were washed with PBS/0.1% Tween 20/10 μg/ml heparin at room temperature for 48 h, with the fresh buffer changed every 8 h. All the incubation steps were performed with gentle rotation.

Techniques: Saline, Enzyme-linked Immunosorbent Assay, Imaging, Immunolabeling, Staining

Journal: Cell Insight

Article Title: Cannabinoid receptor 2 signal promotes type 2 immunity in the lung

doi: 10.1016/j.cellin.2023.100124

Figure Lengend Snippet: Blockage of the CB2 signal ameliorates papain-induced lung pathology. CB2 −/− and control CB2 +/+ littermates were subjected to intranasal papain treatment. (A) mRNA levels of IL-4 , IL-5 , and IL-13 in the lung tissues of CB2 +/+ and CB2 −/− mice were determined by the qPCR analysis and normalized to the saline-treated CB2 +/+ condition. n ​= ​5 for saline-treated condition and n ​= ​16 for papain-treated condition, mean ​± ​SEM, ∗ p < 0.05 (two-way ANOVA test). (B and C) Papain-induced lung inflammation was visualized by advanced 3D imaging. The intact, unsectioned lungs (left lobe) of CB2 +/+ and CB2 −/− mice were processed for the whole-tissue anti-Siglec-F immunolabeling. (B) Representative 3D-projection images at 1.26 × magnification of the lightsheet imaging were shown. (C) The density of Siglec-F + immune cells was quantified. n ​= ​5, mean ​± ​SEM, ∗ p < 0.05 (two-way ANOVA test). (D) Eosinophil recruitment in the lungs of CB2 +/+ and CB2 −/− mice was examined by the FACS analysis. n ​= ​5 for saline-treated condition and n ​= ​10 for papain-treated condition, mean ​± ​SEM, ∗ p < 0.05 (two-way ANOVA test). (E and F) The lung tissues of CB2 +/+ and CB2 −/− mice were assessed by hematoxylin and eosin (H&E) staining. (E) Representative images were shown. (F) Histologic scores were determined. n ​= ​5, mean ​± ​SEM, ∗ p < 0.05 (two-way ANOVA test).

Article Snippet: The tissues were blocked with PBS/0.2% Triton X-100/10% DMSO/5% normal donkey serum at room temperature overnight and then immunolabeled with rat anti-Siglec-F primary antibody (BD Biosciences, Cat#552125, RRID:AB_394340; final concentration of 1 μg/ml) in PBS/0.1% Tween 20/10 μg/ml heparin/5% normal donkey serum at room temperature for 72 h. The tissues were washed with PBS/0.1% Tween 20/10 μg/ml heparin at room temperature for 12 h, with the fresh buffer changed every 2 h. The tissues were further immunolabeled with Alexa Fluor 647-conjugated donkey anti-rat IgG secondary antibody (Thermo Fisher Scientific, Cat#A78947, RRID:AB_2910635; final concentration of 2 μg/ml) in PBS/0.1% Tween 20/10 μg/ml heparin/5% normal donkey serum at room temperature for 72 h. The tissues were washed with PBS/0.1% Tween 20/10 μg/ml heparin at room temperature for 48 h, with the fresh buffer changed every 8 h. All the incubation steps were performed with gentle rotation.

Techniques: Saline, Imaging, Immunolabeling, Staining

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: List of mAb used for the analysis of lymphocyte subsets. BD (BD Biosciences, San Jose, CA); eB (eBioscience, San Diego, CA); DN: double negative; DP: double positive; SP: single positive; T CM : central memory T cell; T EM : effector memory T cell; CD26 expression was assessed in all lymphocyte subsets.

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Expressing, Marker

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: Splenocytes were obtained at 4 and 6 weeks after treatment. A) Percentage of T effector memory (EM) , T central memory (CM) and naïve T lymphocytes (CD8 + and CD4 + ) in control (white circles) and treated mice (black circles). B) Representative FACS plots of the naïve (CD3 + CD4 + /CD8 + CD44 low CD62L + ), T CM (CD3 + CD4 + /CD8 + CD44 high CD62L + ) and T EM (CD3 + CD4 + /CD8 + CD44 high CD62L neg ) both for CD4 + and CD8 + T lymphocytes from spleen in treated and control mice at 4 weeks of treatment. C) MFI for CD26 expression on T EM, T CM and naïve T lymphocytes (CD8 + and CD4 + ) in control (white circles) and treated mice (black circles). Lines represent the mean of 8–10 mice. Comparison between treated and untreated mice showed an increased expression of CD26 on CD8 + T EM cells at any of the study’s time-points. (*p<0.05, **p<0.01, three way Anova).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Control, Expressing, Comparison

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: T cells from PLNs were obtained from control (white circles) and treated (black circles) NOD mice at 4 weeks after treatment. A) Percentage of T effector memory (EM) , T central memory (CM) and naïve T lymphocytes (CD8 + and CD4 + ). B) MFI for CD26 expression on T EM, T CM and naïve T lymphocytes (CD8 + and CD4 + ). The expression of CD26 was significantly increased on CD4 + T EM and CD8 + T EM cells in MK626-treated mice when compared to controls. Lines represent the mean of 6 mice (*p<0.05, ***p<0.001, three way Anova).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Control, Expressing

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: A) Percentage of CD8 + and CD4 + T lymphocytes infiltrating pancreatic islets in untreated (white circles) and treated (black circles) mice assessed by flow cytometry. Lines represent the mean of 6–9 mice. No statistical differences were observed between groups (Mann-Whitney test). B) Expression of CD26 determined by flow cytometry (MFI) in CD8 + and CD4 + T lymphocytes from control (white circles) and treated (black circles) mice. Lines represent the mean of 4–8 mice. (*p≤0.05, Mann-Whitney test).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Flow Cytometry, MANN-WHITNEY, Expressing, Control

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: CD8 + T EM cell from control (n = 9) and treated (n = 9) mice obtained at 4 weeks of treatment were stimulated with anti–CD3 in vitro . A) Proliferative rate in CD8 + T EM lymphocytes from MK626-treated mice (black circles) and in control group (white circles). T cell proliferation was expressed as counts per minute (c.p.m) using 3 H-TdR. B) Cytokine concentration (IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IFN-γ, TNF-α and TGF-β) in supernatant from proliferation assays in treated mice (black bars) compared to control mice (white bars). Results are expressed as mean ± SEM. Mann-Whitney test was used for the evaluation of statistical significance (*p<0.05;**p<0.01).

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: Control, In Vitro, Concentration Assay, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Type 1 Diabetes Prevention in NOD Mice by Targeting DPPIV/CD26 Is Associated with Changes in CD8 + T Effector Memory Subset

doi: 10.1371/journal.pone.0142186

Figure Lengend Snippet: A) Representative dot plots showing CFSE-T lymphocytes, stimulated with insulin-loaded mDCs, in the presence or absence of CTV-CD8 + T EM lymphocytes (ratio 1:1) and MK626 (10μM). B) Percentage of proliferating CFSE low T lymphocytes versus all T lymphocytes, with insulin-loaded mDCs (circles and triangles), in the presence of CTV-CD8 + T EM lymphocytes (triangles) and MK626 (open symbols) for 3 days. Plots show the mean (line) of three independent experiments.

Article Snippet: For the identification of cell subsets, cell suspension was stained with a primary antibody Rat anti-mouse CD8 (clone Lyt-2.2; MyBiosource, San Diego, CA).

Techniques: