mouse ifnγ quantikine elisa  (R&D Systems)

Journal: Journal of Nanobiotechnology

Article Title: XOR-mediated tumor-selective nanomaterial therapy: achieving tumor and immune cell dual regulation through intracellular ROS generation

doi: 10.1186/s12951-026-04150-6

Figure Lengend Snippet: ZIF-8@XOR treatment promotes anti-tumor immune cell infiltration and activation (A) Intracellular cytokines of TAMs, including TNFα, IL6, IL10, and TGFβ in the different treatment groups. (B) Intracellular p-STAT1 and p-STAT6 expression of TAMs in the different treatment groups. (C) Tumor-infiltrating CD8 + and CD4 + T cells in the different treatment groups. (D) Representative flow cytometry analysis of splenetic CD8 + and CD4 + T cells in the different treatment groups. (E) PD1, TIGIT, and LAG3 expression on the surface of tumor-infiltrating CD8 + T cells in the different treatment groups. (F) Intracellular anti-tumor molecules of tumor-infiltrating CD8 + T cells, including Granzyme B, Perforin, IFN-γ, IL2, and TNF-α in the different treatment groups. (G) Expression level of TNF-α and IFN-γ in tumor tissue were analyzed by ELISA. (H) Expression level of (p)PI3K, (p)AKT, (p)mTOR and (p)STAT1 of tumor-infiltrating CD8 + T cells, sorted from tumors at the endpoint of each treatment group. (I) Quantitative analysis of ROS expression in tumor from different treatment groups based on DHE MFI detected by flow cytometry. (J) Pearson correlation analysis of intratumoral ROS levels with TAM M1 polarization and CD8 + T cells tumor infiltration. r > 0 indicates positive correlation. The data are represented as the mean±SD ( n = 3; ns, no significance; * p < 0.05, *** p < 0.001, and **** p < 0.0001)

Article Snippet: Quantification of TNF-α and IFN-γ in the supernatants was performed by mouse TNF-α and IFN-γ ELISA kits (R&D Systems, MN, USA).

Techniques: Activation Assay, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: Engineered Small Extracellular Vesicles Targeting Tumor‐Associated Endothelial Cells to Effectively Remodel the Glioma Microenvironment

doi: 10.1002/advs.202518490

Figure Lengend Snippet: sEVs Carrying cGAMP and Tumor Antigen Induce Strong Tumor Specific Immune Response. 23 days after tumor cell transplantation, mice were sacrificed, brain, spleen and lymph node were removed and analyzed. (A) Immune fluorescence staining analysis of proinflammatory (iNOS, green) and anti‐inflammatory (ARG1, red) immune cells in the tumor. Scale bar=100 µm. (B,C) Quantitative analysis of ARG1 + cells (B) and iNOS + cells (C). n=5 mice per group, one‐way ANOVA. (D) Number of tumor‐infiltrating immune cells. n=5 mice per group, one‐way ANOVA. (E) Elisa analysis of brain immune cells activation. Tumor infiltrated immune cells were isolated by 30/70% Percoll density gradient centrifugation. 5 × 10 5 cells were seeded in 96‐well plate and cultured for 48 h, the concentration of IFN‐γ in each supernatant was analyzed by ELISA. n=3 mice per group, one‐way ANOVA. (F‐M) Flow cytometry analysis of tumor infiltrated myeloid cells (F) and lymphocyte (K). Immune cells isolated from the tumors were treated with PMA (50 ng/ml), ionomycin (500 ng /ml) and GolgiPlug (1 µg/ml) for 4 h, then cells were successively stained with surface and intracellular antibodies, and analyzed by flow cytometry. The percentage of TNF‐α + (G) or IL‐10 + (H) myeloid cells were quantified, and the ratio of TNF‐α + to IL‐10 + myeloid cells was calculated (I). (J) Quantification of MHCII positive myeloid cells. (L, M) The percentages of CD4 + (L) or CD8 + (M) T cells in lymphocytes were quantified. G‐J, L, M, n=5 mice per group, one‐way ANOVA. (N, O) Tumor antigen specific immune response assay. CD8 + T cells were isolated from tumor infiltrated immune cells using CD8a Microbeads, and co‐cultured with dendritic cells pre‐pulsed with PEP‐3 peptide (N) or GL261‐EGFRvIII cell line (O) for 48 h, the release of IFN‐γ was analyzed by ELISA. n=4 mice per group, one‐way ANOVA. (P) Elisa analysis of peripheral immune cells activation. Peripheral immune cells were isolated from the spleen and lymph node of mice. 5 × 10 5 cells were seeded in 96‐well plate and cultured for 48 h, the concentration of IFN‐γ in each supernatant was analyzed by ELISA. n=5 mice per group, one‐way ANOVA. All results are expressed as mean ± SD, * p < 0.05, ** p < 0.01, *** p <0.001, **** p < 0.0001, ns=not significant.

Article Snippet: The supernatants from all cultures were collected, and the secretion of IFN‐γ was analyzed using a commercial mouse IFN‐γ ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Transplantation Assay, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, Activation Assay, Isolation, Gradient Centrifugation, Cell Culture, Concentration Assay, Flow Cytometry

Journal: Advanced Science

Article Title: Engineered Small Extracellular Vesicles Targeting Tumor‐Associated Endothelial Cells to Effectively Remodel the Glioma Microenvironment

doi: 10.1002/advs.202518490

Figure Lengend Snippet: Comparison of the Therapeutic Effects Between i.v. Injected sEVs Carrying cGAMP and Intratumoral Injected Free cGAMP. (A) Schematic diagram of experiment timeline. (B) IVIS imaging evaluation of tumor volume. Tumor volume was monitored every five days from 7 days post tumor cells inoculation. (C) Tumor growth curve. n=5 mice per group, one‐way ANOVA. (D) Histological staining analysis of tumor tissue. Scale bar=100 µm. (E) Immunofluorescence staining analysis of tumor cell apoptosis after different treatments. Scale bar=50 µm. (F) Quantitative analysis of TUNEL positive cells. n=5 mice per group, one‐way ANOVA. (G) Evaluation of STING activation in the tumor. Tumor slices were co‐stained with pSTING (green) and CD31 (red) antibodies to analyze the specificity of STING activation in different groups. Scale bar=50 µm. (H) The number of pSTING‐positive cells and the number of pSTING‐positive cells among the CD31‐positive cells were quantified, and the percentage of pSTING + CD31 + cells relative to the pSTING + cells was calculated. n=5 mice per group, one‐way ANOVA. (I) Flow cytometry analysis of tumor infiltrated CD8 + T cells. Immune cells isolated from the tumors were treated with PMA (50 ng/ml), ionomycin (500 ng /ml) and GolgiPlug (1 µg/ml) for 4 h, then cells were successively stained with surface and intracellular antibodies, and analyzed by flow cytometry. The percentage of CD8 + T cells in lymphocytes (J), IFN‐γ + in CD8 + T cells (K), PD‐1 + in CD8 + T cells (L) were quantified. n=5 mice per group, one‐way ANOVA. (M) Tumor antigen specific immune response assay. CD8 + T cells were isolated from tumor infiltrated immune cells using CD8a Microbeads, and co‐cultured with CT2A‐EGFRvIII cell line for 48 h, the release of IFN‐γ was analyzed by ELISA. n=4 mice per group, one‐way ANOVA. All results are expressed as mean ± SD, a= p < 0.05, b= p < 0.01, ns=not significant.

Article Snippet: The supernatants from all cultures were collected, and the secretion of IFN‐γ was analyzed using a commercial mouse IFN‐γ ELISA kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Comparison, Injection, Imaging, Staining, Immunofluorescence, TUNEL Assay, Activation Assay, Flow Cytometry, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay