mouse ifn-gamma elisa kit  (Multi Sciences (Lianke) Biotech Co Ltd)

Journal: Advanced Science

Article Title: Coixenolide Enhances Antitumor Immunity of Cytotoxic CD8 + T Cells by Inhibiting S100A8/A9‐CD36 Axis in Obesity‐Associated Pancreatic Adenocarcinoma

doi: 10.1002/advs.202515759

Figure Lengend Snippet: Transcriptomic analyses revealed that coixenolide significantly inhibited S100a8/a9 + PAAD cells in HFD‐fed mice. (A) Subclusters of epithelial cells. (B) Malignancy score of epithelial cells. (C) The heterogeneity of epithelial cell clusters among different groups and tissue samples based on cell ratio. (D,E) Changes in the proportions of Cluster 2 (D) and Cluster 8 (E) tumor cells. n = 7 for HFD group and n = 6 for CD group. Data are presented as mean ± SD; a two‐sided t ‐test was used. (F) The upregulated genes across 21 tumor cell subsets. (G) Pathway enrichment associated with the top 100 upregulated genes (ranked by p ‐value) of Clusters 2 and 8 tumor cells. (H) CytoTRACE score and distribution of tumor cell clusters. (I) Shared genes of Clusters 2 and 8. (J) The colocalization of S100A8/A9 (yellow), Pan‐CK (red), and DAPI (deep blue) in the tumor tissues of CD‐ and HFD‐fed mice; representative of three independent experiments. (K) The expression patterns of S100A8 and S100A9 between tumor and normal pancreatic tissues using the GEPIA webtool; n = 179 (tumor tissue) and 171 (normal tissue); a two‐sided Wilcoxon test was used. (L) The colocalization of S100A8/A9 (yellow), insulin (pink), Pan‐CK (white), and DAPI (deep blue) in the peritumoral pancreatic tissue, as well as in tumor tissues in PAAD patients. (M,N) Kaplan–Meier survival plot indicating that patients with high levels of S100A8 (M) or S100A9 (N) have a worse clinical outcome in the TCGA PAAD cohort; log‐rank test was used. (O,P) The expressions of S100A8 (O) and S100A9 (P) between patients with PAAD of different invasion levels, grades, and AJCC stages. The data were sourced from the TCGA cohort; a two‐sided t ‐test was used. (Q) Expression levels of S100a8 and S100a9 in Clusters 2 and 8 tumors of HFD‐fed mice following coixenolide treatment. A two‐sided t ‐test was used. (R,S) Western blot (R) and flow cytometry plot (S) measuring S100a8/a9 expression in Pan02 cells following coixenolide treatment. n = 4 independent experiments; two‐sided t ‐test was used. (T) The levels of S100a8/a9 in the supernatants of Pan02 cells with or without coixenolide treatment were determined using ELISA. n = 3 independent experiments; two‐sided t ‐test was used. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The CD8 + T cell supernatants were collected, and the secretion of TNF‐α (Mouse TNF‐alpha ELISA Kit, RK00027, Abclonal), IFN‐γ (Mouse IFN‐gamma ELISA Kit, RK00019, Abclonal), and GZMB (Mouse Granzymes B ELISA Kit, RK00007, Abclonal) was quantified using specific ELISA kits following the standard protocols.

Techniques: Expressing, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Coixenolide Enhances Antitumor Immunity of Cytotoxic CD8 + T Cells by Inhibiting S100A8/A9‐CD36 Axis in Obesity‐Associated Pancreatic Adenocarcinoma

doi: 10.1002/advs.202515759

Figure Lengend Snippet: Cell–cell communication analysis indicated that S100a8/a9 served as a critical target for coixenolide, regulating the interactions between PAAD cells and CD8 + T cells. (A) The overview of cell–cell interactions between malignant epithelial cells and T cells in the tumor tissues treated with vehicle control and coixenolide, respectively. (B) The intensity of intercellular signaling, showing both the sent and received signals. The cells in the upper right corner exhibit the strongest incoming and outgoing interactions. S100a8/a9 + tumor cell clusters (Clusters 2 and 8) are highlighted in red boxes. Cytotoxic CD8 + T and Treg cells are indicated by red arrows. (C) The outgoing communication patterns of epithelial cells and T cells in the tumor of HFD‐fed mice with or without coixenolide treatment. (D) CD8 + T cell killing assays were conducted using coculture of Pan02 and CD8 + T cells. Pan02 or CD8 + T cells were pretreated with 25 mg/mL coixenolide before coculture. (E) Primary CD8 + T cells were activated by incubation with CD3/CD28 antibody and labeled with carboxy‐fluorescein succinimidyl ester (CFSE). (F–H) The levels of TNF‐α (F), IFN‐γ (G), and Granzyme B (GZMB) (H) in the supernatants after co‐culture of CD8 + T and Pan02 cells pretreated with coixenolide (+) or vehicle control (−) were determined using ELISA. (I–K) The expression of TNF‐α (I), IFN‐γ (J), and Granzyme B (GZMB) (K) in CD8 + T cells after co‐culture of CD8 + T cells and Pan02 cells pretreated with coixenolide (+) or vehicle control (−) was determined using flow cytometry. (L) Flow cytometry analyses of T cell exhaustion markers (CTLA‐4) on CD8 + T cells after coculture of Pan02 and CD8 + T cells pretreated with coixenolide (+) or vehicle control (−). (M,N) Flow cytometry analyses of CD8 + T cell killing (M) and division (N) activities. CD8 + T cells were cocultured with S100a9 knockout (KO) or wild‐type (WT) Pan02 cells. (O–Q) The levels of TNF‐α (O), IFN‐γ (P), and GZMB (Q) in the supernatants after coculture of CD8 + T cells with WT or S100a9 KO Pan02 cells were determined using ELISA. n = 3 or 4 independent coculture experiments or replicates. One‐way ANOVA with Tukey's post‐hoc test (E–L), two‐sided t ‐test (M–N), or two‐way ANOVA (O‐Q) was used. All p values are two‐sided. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The CD8 + T cell supernatants were collected, and the secretion of TNF‐α (Mouse TNF‐alpha ELISA Kit, RK00027, Abclonal), IFN‐γ (Mouse IFN‐gamma ELISA Kit, RK00019, Abclonal), and GZMB (Mouse Granzymes B ELISA Kit, RK00007, Abclonal) was quantified using specific ELISA kits following the standard protocols.

Techniques: Control, Incubation, Labeling, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Knock-Out

Journal: Cancer Research

Article Title: Soluble Uric Acid Drives CD8 + T-cell Exhaustion by Inducing KSR1-Mediated MAPK Hyperactivation

doi: 10.1158/0008-5472.CAN-25-3911

Figure Lengend Snippet: UA directly induces CD8 + T-cell exhaustion and impairs cytotoxicity. A, Representative flow cytometry plots showing IFNγ, GZMB, PD-1, and Tim-3 expression in OT-1 CD8 + T cells after 7 days of in vitro stimulation with 100 ng/mL OVA peptide, with or without UA treatment (70 μg/mL). B, Quantification of IFNγ, GZMB, PD-1, and Tim-3 expression levels corresponding to A . C, In vitro cytotoxicity assay assessing the killing efficiency of OT-1 CD8 + T cells cultured for 7 days with or without UA against CFSE-labeled MC38-OVA target cells at E:T ratios of 1:1, 2:1, 4:1, and 8:1. Target cell apoptosis was measured by active caspase-3 expression. Tim-3 expression on CD8 + T cells under each condition was also analyzed by flow cytometry. D, Quantitative analysis of killing efficiency and Tim-3 expression from C . E, qPCR analysis of Ifng and Gzmb mRNA expression in OT-1 CD8 + T cells cultured with or without UA on days 5 and 7 after activation. F, ELISA quantification of IFNγ and GZMB protein levels in culture supernatants from OT-1 CD8 + T cells with or without UA treatment, measured on days 5 and 7 after activation. G, Schematic of the in vivo experimental model: CD45.1 + C57BL/6J mice bearing subcutaneous MC38-OVA tumors received intratumoral adoptive transfer of CD45.2 + WT or Havcr2 −/− OT-1 CD8 + T cells pretreated with or without UA ( n = 5 mice per group). H, Tumor growth curves of MC38-OVA tumors following adoptive transfer of the indicated CD8 + T-cell groups described in G . I, Final tumor weights at endpoint for each group described in G . J, Flow cytometry analysis of functional markers (IFNγ and GZMB) and exhaustion markers (PD-1 and Tim-3) in tumor-infiltrating CD45.2 + WT or Havcr2 −/− OT-1 CD8 + T cells from experiments described in G . I and J, share the same legend and color codes as H . Note: for I and J , n = 3 in the Havcr2 −/− OT-1 CD8 + T group, as analysis was restricted to residual tumors; other mice achieved complete remission. Statistical significance was determined by comparison of endpoint tumor volumes/weights. Data represent mean ± SEM. Statistical significance was determined by one-way ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Mouse IFNγ and GZMB concentrations in supernatants were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (ABclonal) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Expressing, In Vitro, Cytotoxicity Assay, Cell Culture, Labeling, Activation Assay, Enzyme-linked Immunosorbent Assay, In Vivo, Adoptive Transfer Assay, Functional Assay, Comparison