rabbit anti-human nos2  (Santa Cruz Biotechnology)

Journal: Oncoimmunology

Article Title: Nitric oxide synthase 2 is involved in the pro-tumorigenic potential of γδ17 T cells in melanoma

doi: 10.1080/2162402X.2016.1208878

Figure Lengend Snippet: NOS2 deficiency delays melanoma progression by inhibiting tumor cell dissemination (A–C) 6-mo follow-up of melanoma development from Ret (n = 40) and RetNos2KO (n = 40) mice. (A,B) Time courses of primary tumor and cutaneous metastasis onset. Mice were examined every 2 weeks. (C) Survival curve. (Mantel-Cox test (A–C)). (D, E) trp2 expression was measured by qRT-PCR in bladders (D) and tumor dLNs (E) collected from Ret and RetNos2KO mice aged of 2, 5 or 12 weeks. Expressions were normalized using GAPDH. Each point represents one mouse. (F) Number of pulmonary metastasis in WT mice and Nos2KO mice after i.v. injections of 105 (n = 8 WT, n = 9 Nos2KO) or 106 (n = 6 per group) B16 melanoma cells. Data are pooled from two independent experiments (mean ± SEM) (Mann–Whitney test). *p < 0.05, **p < 0.01.

Article Snippet: Microscopy was performed using primary Abs; rabbit anti-human NOS2 (SP126, ThermoFisher Scientific), mouse anti-human γδ TCR (11F2, BD Biosciences), biotin hamster anti-mouse γδ TCR (GL3, BD Biosciences) and rabbit anti-mouse NOS2 (Calbiochem).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Nitric oxide synthase 2 is involved in the pro-tumorigenic potential of γδ17 T cells in melanoma

doi: 10.1080/2162402X.2016.1208878

Figure Lengend Snippet: NOS2 deficiency reduces PMN-MDSCs infiltration in primary tumors (A) Protein levels of indicated cytokines in primary tumors, from Ret (n = 14, except for G-CSF n = 11, IL-17 n = 10 and VEGF n = 8) and RetNos2KO (n = 8, except for IL-17 n = 6) mice, determined by ELISA and multiplex ELISA. (B) Proportions of myeloid cells among CD45+ cells within primary tumors of Ret (n = 35) and RetNos2KO (n = 16) mice (C,D) Dendritic cells (DC: CD11c+), macrophages (MФ: CD11b+CD11c-Ly6C−/lowLy6G-), monocytic MDSCs (M-MDSCs: CD11b+CD11c−Ly6ChighLy6G−) and PMN-MDSCs (CD11b+CD11c−Ly6ClowLy6G+) were analyzed by flow cytometry. Percentages (C) and absolute numbers (D) of myeloid cell subsets within primary tumors of Ret (n = 35) and RetNos2KO (n = 16) mice are shown (left). Representative dot plots of PMN-MDSCs staining (right), Data are pooled from at least three independent experiments. Each point represents individual mouse. Bars are mean ± SEM. *p < 0.05, **p < 0.01 ***p < 0.001 (Mann–Whitney test).

Article Snippet: Microscopy was performed using primary Abs; rabbit anti-human NOS2 (SP126, ThermoFisher Scientific), mouse anti-human γδ TCR (11F2, BD Biosciences), biotin hamster anti-mouse γδ TCR (GL3, BD Biosciences) and rabbit anti-mouse NOS2 (Calbiochem).

Techniques: Enzyme-linked Immunosorbent Assay, Multiplex Assay, Flow Cytometry, Staining, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Nitric oxide synthase 2 is involved in the pro-tumorigenic potential of γδ17 T cells in melanoma

doi: 10.1080/2162402X.2016.1208878

Figure Lengend Snippet: NOS2 deficiency decreases IL-17-producing γδ Tcells (A) Percentage of γδ T cells among CD45+ cells in primary tumors of Ret (n = 9) and RetNos2KO mice (n = 11). (B) Correlation between the numbers of γδ T cells and PMN-MDSCs within the primary tumors of Ret (n = 33) (left) and RetNos2KO (n = 17) (right) mice. Each point represents individual mouse and line is linear regression. (C–E) IL-17 production assessed by flow cytometry on single-cell suspensions derived from primary tumors of Ret (n = 11) and RetNos2KO mice (n = 10) after PMA/ionomycin stimulation. (C) Percentage of total IL-17producing CD45+ cells. (D) Representative FSC/IL-17 dot plots are shown for γδ T cells and CD4+ T cells. (E) Percentage (left) and absolute number (right) of γδ T cells and CD4+ T cells producing IL-17. Bars are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (Mann–Whitney test A, C–E and Pearson correlation test B).

Article Snippet: Microscopy was performed using primary Abs; rabbit anti-human NOS2 (SP126, ThermoFisher Scientific), mouse anti-human γδ TCR (11F2, BD Biosciences), biotin hamster anti-mouse γδ TCR (GL3, BD Biosciences) and rabbit anti-mouse NOS2 (Calbiochem).

Techniques: Flow Cytometry, Derivative Assay, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Nitric oxide synthase 2 is involved in the pro-tumorigenic potential of γδ17 T cells in melanoma

doi: 10.1080/2162402X.2016.1208878

Figure Lengend Snippet: NOS2 improves pro-tumor properties of γδ Tcells. (A, B) Cytotoxic activity of γδ T cells derived from Ret and RetNos2KO mice. B16 cells were seeded in wells of E-plates. γδ T cells stimulated with anti-CD28 (A) or anti-CD3 plus anti-CD28 (B) were added to a ratio of one γδ T cell for two B16 cells (see Methods). The inhibition of tumor cell proliferation was assessed by dynamically monitoring every 30 min on cell impedance. The graph (left) shows the normalized cell index values. The percent of B16 melanoma cell lysis (right) was calculated 4 h after addition of effector cells. Data are pooled from two independent experiments with 9 (non-stimulated and stimulated Ret), 11 (stimulated RetNos2KO) and 13 (non-stimulated RetNos2KO) replicates. (C,D) IFNγ production assessed by flow cytometry on single-cell suspensions derived from primary tumors of Ret (n = 5) and RetNos2KO mice (n = 4) after PMA/ionomycin stimulation. (C) Percentage of total IFNγ producing CD45+ cells. (D) Percentage (left) and absolute number (right) of γδ T cells producing IFNγ. (E,F) 4-mo follow-up of melanoma development from Ret (n = 37) and RetγδKO (n = 26) mice. Time courses of primary tumor (F) and cutaneous metastasis (F) onset. Mice were examined every 2 weeks. Data are mean ± SEM. ((A)and (B) left, C,D) except mean ((A)and (B) right). Each point represents one replicate ((A)and (B) right). *p < 0.05, ***p < 0.001 (Mann–Whitney test (A–C) and Mantel-Cox test (E,F)).

Article Snippet: Microscopy was performed using primary Abs; rabbit anti-human NOS2 (SP126, ThermoFisher Scientific), mouse anti-human γδ TCR (11F2, BD Biosciences), biotin hamster anti-mouse γδ TCR (GL3, BD Biosciences) and rabbit anti-mouse NOS2 (Calbiochem).

Techniques: Activity Assay, Derivative Assay, Inhibition, Lysis, Flow Cytometry, MANN-WHITNEY

Journal: Oncoimmunology

Article Title: Nitric oxide synthase 2 is involved in the pro-tumorigenic potential of γδ17 T cells in melanoma

doi: 10.1080/2162402X.2016.1208878

Figure Lengend Snippet: γδ Tcells infiltrating mouse and human melanoma are able to express NOS2. (A) Representative confocal microscopy images showing γδ T cells positive for NOS2 from cells derived from TdLNs of Ret mice and stained with antibodies to TCR γδ (red), NOS2 (green) and counterstained with DAPI (blue). Bars 10 µM. 40 X objective (left). Quantification of γδ T cells and γδ T cells positive for NOS2 from 500 to 1,500 cells (right). Experiments were performed five times. (B) Representative digital microscopy images of 7 µm sections from frozen biopsies of human primary melanoma (patient 14 and 12, respectively). Sections were stained with antibodies to TCR γδ (red) and NOS2 (green). Nuclei were counterstained with DAPI (blue). Bars 5 µM. 40 X objective (left). Quantification of γδ T cells and γδ T cells positive for NOS2 in 13 patients with detected γδ T cells (right). Patients were divided into three groups with low, intermediate or high risk of disease's relapse according to Breslow thickness. Arrows indicate NOS2-expressing γδ T cells.

Article Snippet: Microscopy was performed using primary Abs; rabbit anti-human NOS2 (SP126, ThermoFisher Scientific), mouse anti-human γδ TCR (11F2, BD Biosciences), biotin hamster anti-mouse γδ TCR (GL3, BD Biosciences) and rabbit anti-mouse NOS2 (Calbiochem).

Techniques: Confocal Microscopy, Derivative Assay, Staining, Microscopy, Expressing