Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type I IFN-producing CD4 Valpha14i NKT cells facilitate priming of IL-10-producing CD8 T cells by hepatocytes.

doi: 10.4049/jimmunol.178.4.2083

Figure Lengend Snippet: FIGURE 6. The cytokine response of OT-I CD8 T cells primed by HC with or without NKT cell help. A, OT-I CD8 T cells (105/well) were cocul- tured with GalCer- and SIINFEKL peptide-pulsed HC (104/well) with () or without () NKT cells (4 104/well). Cells were harvested after a 72-h culture, incubated for 4 h with ionomycin (750 ng/ml) and PMA (50 ng/ml), washed, and tested for IL-10 secretion in a 3-h capture assay. Cells were labeled with a PE-conjugated anti-IL-10 mAb and an anti-CD8 mAb. Data from a representative of five independent experiments are shown. B, OT-I CD8 T cells (105/well) were cocultured for 72 h with GalCer-/ SIINFEKL peptide-pulsed HC (104/well) with () or without () NKT cells (4 104/well). CD8 T blasts were harvested, purified, and restimu- lated in vitro for 48 h with peptide-pulsed DC. IFN-, IL-2, and IL-10 in 48-h supernatants of these secondary cultures were determined by ELISA. Mean values of triplicates (SEM) of a representative of four independent experiments are shown.

Article Snippet: IL-10-producing CD8 T cells were identified by a cytokine capture assay (catalog no. 130-090-489; Miltenyi Biotec) combined with surface staining with the allophycocyanin-conjugated anti-CD8 mAb 53-6.7 (catalog no. 553035; BD Biosciences).

Techniques: Incubation, Labeling, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Medicine

Article Title: miR-1 suppresses the growth of esophageal squamous cell carcinoma in vivo and in vitro through the downregulation of MET, cyclin D1 and CDK4 expression

doi: 10.3892/ijmm.2016.2619

Figure Lengend Snippet: Effect of miR-1 overexpression/downregulation on apoptosis. (A and B) Flow cytometric analysis of apoptosis following transfection with miR-1 mimics, inhibitor and their respective NCs. (C) Rate of apoptosis was determined following transfection with miR-1 mimics, inhibitor and their respective NCs. * P<0.05.

Article Snippet: The rate of apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis detection kit (Beyotime Institute of Biotechnology, Shanghai, China).

Techniques: Over Expression, Transfection

Journal: Molecular medicine reports

Article Title: Upregulation of miR-142-5p in atherosclerotic plaques and regulation of oxidized low-density lipoprotein-induced apoptosis in macrophages.

doi: 10.3892/mmr.2015.3191

Figure Lengend Snippet: Figure 3. (A) MicroRNA (miR)‑142‑5p inhibited apoptosis of macrophages. The macrophages were transfected with a miR‑142‑5p inhibitor or mimic and/or transforming growth factor β2 (TGF‑β2) inhibitor, and incubated with oxidized low‑density lipoprotein (ox‑LDL) for 24 h. Red colouration indicates apoptosis. A, Annexin V‑phycoerythrin staining of red fluorescent map; B, macrophages in situ; C, A and B combined. (B) Quantification of the percentage of apoptotic cells. The results are expressed as the means ± standard error of the mean. *P<0.05 vs control, #P<0.05 vs control+ox‑LDL.

Article Snippet: The number of apoptotic human macrophages was quantified using the Annexin V-PE Apoptosis Detection kit (Beyotime Institute of Biotechnology, Hainen, China).

Techniques: Transfection, Incubation, Staining, In Situ, Control

Journal: Cell Death & Disease

Article Title: Urocortin protects chondrocytes from NO-induced apoptosis: a future therapy for osteoarthritis?

doi: 10.1038/cddis.2013.231

Figure Lengend Snippet: Apoptotic and necrotic chondrocyte cell death assessed as the percentage of Annexin V- and TUNEL-positive cells (apoptosis) and cellular LDH release (necrosis) following treatment of C-20/A4 cells with increasing concentrations of SNAP ( a ) and TNF- α ( b ). * P <0.05. ** P <0.01 compared with control (untreated)

Article Snippet: Apoptosis was assessed by fluorescence microscopy by Annexin V binding (TACS Annexin V-FITC kit, R&D systems) and by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL; in situ cell death detection kit, Roche diagnostics, Lewes, UK).

Techniques: TUNEL Assay, Control

Journal: Cell Death & Disease

Article Title: Urocortin protects chondrocytes from NO-induced apoptosis: a future therapy for osteoarthritis?

doi: 10.1038/cddis.2013.231

Figure Lengend Snippet: Cytoprotective effects of endogenous Ucn. Apoptotic and necrotic chondrocyte cell death assessed as the percentage of Annexin V- and TUNEL-positive cells (apoptosis) and cellular LDH release (necrosis) following the treatment of C-20/A4 cells, with the CRH receptor antagonist α -helical CRH, anti-UCN antibody and anti-albumin antibody, ** P <0.01 compared with control

Article Snippet: Apoptosis was assessed by fluorescence microscopy by Annexin V binding (TACS Annexin V-FITC kit, R&D systems) and by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL; in situ cell death detection kit, Roche diagnostics, Lewes, UK).

Techniques: TUNEL Assay, Control

Journal: Cell Death & Disease

Article Title: Urocortin protects chondrocytes from NO-induced apoptosis: a future therapy for osteoarthritis?

doi: 10.1038/cddis.2013.231

Figure Lengend Snippet: Cytoprotective effects of exogenous Ucn. Apoptotic and necrotic chondrocyte cell death assessed as the percentage of Annexin V- and TUNEL-positive cells (apoptosis) and cellular LDH release (necrosis) following treatment of C-20/A4 cells with Ucn (U) administered concurrently with SNAP (S) or TNF- α (T) in the presence or absence of α -helical CRH (A) * P <0.05, ** P <0.01 compared with control (C).

Article Snippet: Apoptosis was assessed by fluorescence microscopy by Annexin V binding (TACS Annexin V-FITC kit, R&D systems) and by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL; in situ cell death detection kit, Roche diagnostics, Lewes, UK).

Techniques: TUNEL Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Identification and analysis of islet antigen specific CD8+ T cells with T cell libraries

doi: 10.4049/jimmunol.1800267

Figure Lengend Snippet: Libraries were prepared from CD8+CD45RO+ cells sorted from the peripheral blood as described from 29 patients with T1D and 15 HC. Each well was expanded with irradiated allogeneic PBMC+IL-2, IL-7, and IL-15 as described in Materials and Methods. After 10 days the wells were washed and stimulated with K562 cells that had been pulsed with 6 diabetes peptides, peptides from EBV/flu, or treated with DMSO alone. IFNγ levels in the supernatants were measured by ELISA after 6 days. Data from a representative patient and HC subject are shown in Supplemental Figure 2B. The frequencies of positive wells that were above the threshold, which was mean+3SD of DMSO wells were calculated for each subject. There was a significantly greater proportion of positive wells from CD45RO+ cells from patients with T1D reactive with islet antigens (A)(*p=0.028, t-test with Welch’s correction) compared to HC, but not to peptides from EBV/flu (B). CD45RA+CD8+ cells were sorted from 25 and 13 of the T1D patients and HC respectively. (C,D) There was not a significant difference between the frequency of islet antigen-reactive (C) or EBV/flu reactive cells (D). The relationship between frequency of CD45RO+ CD8+ islet antigen-reactive cells and diabetes duration (E) or age (F) are shown (p=ns, Spearman corr). The orange and green dots represent samples from concordant identical triplets and twins respectively.

Article Snippet: To identify the TCRs ex vivo , PBMCs were stimulated with the ZnT8 186–194 peptide for 6 hours and IFNγ+ and – CD8+ cells, identified with anti-CD8 mAb(HIT8a, PerCP-Cy5.5, Biolegend) and an anti-IFNγ antibody (130–054–201, Miltenyi), were sorted by a flow cytometer.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Identification and analysis of islet antigen specific CD8+ T cells with T cell libraries

doi: 10.4049/jimmunol.1800267

Figure Lengend Snippet: (A) Positive wells from the CD8+ T cell libraries from 4 patients with T1D and 3 HC subjects were further expanded with cytokines and challenged with K562 cells pulsed with each individual peptide used in the original pool. The levels of IFNγ were measured after 6 days. The data are from 11 wells from the 3 patients described in the text (Pt 116, Pt 69, and Pt 63) with T1D and 3 HC subjects (HC1023, 19, PC24). Each graph represents the analysis of positive wells from an individual patient. The bars (black and grey) represent the cytokine responses of different positive wells to the peptides. There were positive responses to ZnT8186–194 in wells from the patients with T1D but responses to IGRP228–236, PPI34–42, PPI15–24, and ZnT8186–194 in the HC subjects. (B) The CD45RO+ cells from one library well without and two library wells with an IFNγ response to the peptide pulsed K562 cells from Pt 63 were expanded in cytokines and stained with tetramers loaded with ZnT8186–194 peptide or control tetramer and analyzed by flow cytometry. The percentages refer to the frequency of tetramer+ cells in the CD8+ gate.

Article Snippet: To identify the TCRs ex vivo , PBMCs were stimulated with the ZnT8 186–194 peptide for 6 hours and IFNγ+ and – CD8+ cells, identified with anti-CD8 mAb(HIT8a, PerCP-Cy5.5, Biolegend) and an anti-IFNγ antibody (130–054–201, Miltenyi), were sorted by a flow cytometer.

Techniques: Staining, Control, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Identification and analysis of islet antigen specific CD8+ T cells with T cell libraries

doi: 10.4049/jimmunol.1800267

Figure Lengend Snippet: (A) The Vα and Vβ sequences, identified in 5C above from pt 63, were detected, using PCR for the TCR chains, in positive (+) but not negative (−) library wells (selected for IFNγ responses). (DW=control well) There were multiple wells in the CD45RO libraries but a single positive well from the CD45RA+ libraries in which the Vα and Vβ sequences were identified. (B) PBMC isolated in a repeat draw from the same patient were cultured with ZnT8186–194 peptide. IFNγ+CD8+ and IFNγ−CD8+ T cells were identified with a capture assay and sorted. The presence of the TCR Vα and Vβ chains were again identified by PCR (arrow) in the IFNγ+ cells.

Article Snippet: To identify the TCRs ex vivo , PBMCs were stimulated with the ZnT8 186–194 peptide for 6 hours and IFNγ+ and – CD8+ cells, identified with anti-CD8 mAb(HIT8a, PerCP-Cy5.5, Biolegend) and an anti-IFNγ antibody (130–054–201, Miltenyi), were sorted by a flow cytometer.

Techniques: Control, Isolation, Cell Culture