mouse ifn γ elispot kit  (R&D Systems)

Journal: Poultry Science

Article Title: A novel self-amplified RNA vaccine co-expressing NA and HA1 delivered by Salmonella confers potent protection against H9N2 influenza in chickens

doi: 10.1016/j.psj.2026.107072

Figure Lengend Snippet: Intracellular cytokine production. Chicken splenic lymphocytes were isolated for analysis. Cell proliferation was assessed using CCK-8 analysis with ConA (A), mixed HA1 peptides (B), and NA protein (C). Additionally, the production of IFN-γ by splenic T lymphocytes was measured via an ELISpot assay, utilizing NA and HA1 proteins as stimulators for 36 h (D).

Article Snippet: IFN-γ production was assessed using a commercial Chicken IFN-γ ELISpot kit (Mabtech, Sweden).

Techniques: Isolation, CCK-8 Assay, Enzyme-linked Immunospot

Journal: Poultry Science

Article Title: A novel self-amplified RNA vaccine co-expressing NA and HA1 delivered by Salmonella confers potent protection against H9N2 influenza in chickens

doi: 10.1016/j.psj.2026.107072

Figure Lengend Snippet: Intracellular cytokine production. The intracellular mRNA expression levels of IL-4 (B, D) and IFN-γ (A, C)—as well as the relative concentrations of these cytokines in cell culture supernatants stimulated by the NA peptide (E, F) or HA1 protein (G, H) for 48 h—were determined using qRT-PCR and ELISA, respectively. Data are expressed as the mean ± SEM and analyzed using one-way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.001; n = 4).

Article Snippet: IFN-γ production was assessed using a commercial Chicken IFN-γ ELISpot kit (Mabtech, Sweden).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Human Mutation

Article Title: Whole‐Exome Sequencing to Screen Personal Neoantigens With High Immunogenicity in Patients With Microsatellite Stability (MSS)–Advanced Colorectal Cancer

doi: 10.1155/humu/3876230

Figure Lengend Snippet: Evaluating the immunogenicity of neoantigens from patients with CRC. Autologous PBMCs were stimulated with candidate mutated peptides every 3 days in the presence of IL‐2. On Day 10, T‐cell responses to each antigen were measured by an IFN‐ γ ELISpot assay. The PBMCs were obtained from P1 with CRC. No peptide (medium only) or VSV‐NP 43-69 (STKVALNDLRAYVYQGIKSGNPSILHI) stimulation was used as a control. Data are presented as mean ± SD of three independent experiments. ∗ p < 0.05 and ∗∗ p < 0.01 compared with IFN‐ γ production by PBMCs stimulated without peptide or with VSV‐NP 43-69 .

Article Snippet: After 10 days, the IFN‐ γ response from the prestimulated T cells against neoantigens was assessed using ELISpot assays with a Human IFN‐ γ ELISpot kit (Mabtech).

Techniques: Immunopeptidomics, Enzyme-linked Immunospot, Control

Journal: Human Mutation

Article Title: Whole‐Exome Sequencing to Screen Personal Neoantigens With High Immunogenicity in Patients With Microsatellite Stability (MSS)–Advanced Colorectal Cancer

doi: 10.1155/humu/3876230

Figure Lengend Snippet: Cytotoxicity of NRTs raised by in vitro stimulation of PBLs of P1. NRTs were induced with autologous ZNF169‐A275S– and CDH4‐V456M–pulsed DCs derived from the PBLs of P1. On Day 7, after the third stimulation, the NRTs were harvested for analysis. (A) IFN‐ γ secretion by NRT lines in response to mutated and WT peptides. IFN‐ γ ‐positive SFCs/10 5 NRTs were detected by cytokine‐specific ELISpot. (B–F) Cytotoxicity at the indicated E:T ratios measured by a CCK‐8 kit. Peptide‐specific targets were mutated protein–pulsed T2 cells and minimally nucleated SW480 cells, whereas VSV‐NP 43-69 –pulsed T2 cells, T2 cells alone, and SW480 cells alone were used as controls. Data are expressed as mean ± SEM ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001).

Article Snippet: After 10 days, the IFN‐ γ response from the prestimulated T cells against neoantigens was assessed using ELISpot assays with a Human IFN‐ γ ELISpot kit (Mabtech).

Techniques: In Vitro, Derivative Assay, Enzyme-linked Immunospot, CCK-8 Assay

Journal: Human Mutation

Article Title: Whole‐Exome Sequencing to Screen Personal Neoantigens With High Immunogenicity in Patients With Microsatellite Stability (MSS)–Advanced Colorectal Cancer

doi: 10.1155/humu/3876230

Figure Lengend Snippet: Evaluating the immunogenicity of neoantigens from patients with CRC. Autologous PBMCs were stimulated with candidate mutated peptides every 3 days in the presence of IL‐2. On Day 10, T‐cell responses to each antigen were measured by an IFN‐ γ ELISpot assay. The PBMCs were obtained from P1 with CRC. No peptide (medium only) or VSV‐NP 43-69 (STKVALNDLRAYVYQGIKSGNPSILHI) stimulation was used as a control. Data are presented as mean ± SD of three independent experiments. ∗ p < 0.05 and ∗∗ p < 0.01 compared with IFN‐ γ production by PBMCs stimulated without peptide or with VSV‐NP 43-69 .

Article Snippet: IFN‐ γ in neoantigen‐specific T cells was detected using an ELISpot kit (Mabtech) [ ].

Techniques: Immunopeptidomics, Enzyme-linked Immunospot, Control

Journal: Human Mutation

Article Title: Whole‐Exome Sequencing to Screen Personal Neoantigens With High Immunogenicity in Patients With Microsatellite Stability (MSS)–Advanced Colorectal Cancer

doi: 10.1155/humu/3876230

Figure Lengend Snippet: Cytotoxicity of NRTs raised by in vitro stimulation of PBLs of P1. NRTs were induced with autologous ZNF169‐A275S– and CDH4‐V456M–pulsed DCs derived from the PBLs of P1. On Day 7, after the third stimulation, the NRTs were harvested for analysis. (A) IFN‐ γ secretion by NRT lines in response to mutated and WT peptides. IFN‐ γ ‐positive SFCs/10 5 NRTs were detected by cytokine‐specific ELISpot. (B–F) Cytotoxicity at the indicated E:T ratios measured by a CCK‐8 kit. Peptide‐specific targets were mutated protein–pulsed T2 cells and minimally nucleated SW480 cells, whereas VSV‐NP 43-69 –pulsed T2 cells, T2 cells alone, and SW480 cells alone were used as controls. Data are expressed as mean ± SEM ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001).

Article Snippet: IFN‐ γ in neoantigen‐specific T cells was detected using an ELISpot kit (Mabtech) [ ].

Techniques: In Vitro, Derivative Assay, Enzyme-linked Immunospot, CCK-8 Assay

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-expressing macrophages is associated with response to αOX40 therapy (A) UMAP of monocytes/macrophages subclusters from scRNA-seq data in αOX40-treated MC38-bearing mice. (B) Representative marker genes in the monocyte/macrophage subclusters. (C) Pie chart showing the proportional distribution of monocyte/macrophage subsets of responders and nonresponders. (D) QuSAGE pathway analysis demonstrated enrichment of innate immune and phagocytic signaling pathways in distinct monocyte/macrophage subsets. (E) UMAP showing Mac_C1 signature genes and a heatmap of immune-related gene expression across TAM subclusters ( Z score normalized). (F) Violin plots comparing Nos2 expression levels in Mac_C1 subset between responsive and nonresponsive. (G) Flow cytometry analysis shows the percentage of M1-like (F4/80 + NOS2 + ) and M2-like (F4/80 + CD206 + ) macrophages in tumor tissues of control ( n = 5 mice), nonresponders (with minimal to no response, n = 4 mice), and responders (with a robust therapeutic response, n = 4 mice). (H and I) Comparison of Nos2 expression levels in responders versus nonresponders pre- or post-αOX40 treatment. Bilateral-MC38-bearing mice were treated with αOX40, and tumors from one side were analyzed by RNA-seq prior to (H) or following αOX40 treatment (I). The Nos2 expression was analyzed from RNA-seq data (left) and validated by RT-qPCR (right) ( n = 5 biological replicates). (J) NOS2 expression in tumor biopsies post-treatment determined by RNA-seq. Patients with advanced solid tumors and >1 prior therapy received HFB301001 monotherapy. Tumor biopsy samples were obtained on day 8 of cycle 2 for subsequent RNA-seq analysis. NOS2 expression were compared between patients achieving stable disease (SD, n = 3) and those with progressive disease (PD, n = 3). (K) GO enrichment analysis of upregulated genes in Mac_C1 of responders. (L) Calreticulin expression was quantified by flow cytometry in different response groups following αOX40 treatment ( n = 3 mice per group). (M) NOS2 expression in BMDMs was analyzed by flow cytometry after stimulation with CD8 + T cell supernatant and MC38 lysate, combined with TLR inhibition and IFN-γ blockade ( n = 5 biological replicates). (N) Quantification of Nos2 expression in BMDM by RT-qPCR after 24-h stimulation with MPLA (TLR4 agonist, 100 ng/mL), IFN-γ (20 ng/mL), or both. Data normalized to Gapdh and presented as fold-change relative to unstimulated controls ( n = 4 biological replicates). Data are shown as means ± SD from one of two independent experiments (G, H, I, L, M, and N). Statistical significance was determined using one-way ANOVA with multiple comparisons (G, L, M, and N) or using an unpaired two-tailed t test (H, I, and J). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; VST, variance stabilized transformation; sup., supernatant; lys., tumor lysate; inh., inhibitor.

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Expressing, Marker, Protein-Protein interactions, Gene Expression, Flow Cytometry, Control, Clinical Proteomics, Comparison, RNA Sequencing, Quantitative RT-PCR, Inhibition, Two Tailed Test, Transformation Assay

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: Rational modulation of tumor microenvironment enhances therapeutic responsiveness to αOX40-based immunotherapy (A–D) OX40-humanized mice bearing subcutaneous MC38 (A), B16 (B), E.G7 (C), or KPC (D) tumors ( n = 5–7 mice per group). Tumor growth curves (numbers indicate complete cures) and Kaplan-Meier survival for each model. Treatments: MPLA+IFN-γ ( i.t. , intratumoral); Combo: MPLA+IFN-γ ( i.t. , intratumoral) + αOX40 ( i.p. , intraperitoneal). (E) Study schema of secondary tumor challenge in MC38 model treated with Combo. (F) Tumor progression and survival outcomes following secondary tumor challenge. Growth kinetics of re-implanted tumors in tumor-cleared mice (previously cured by therapy) versus treatment-naive wild-type controls (left). Kaplan-Meier survival plot (right) ( n = 13 mice per group). (G) Systemic immunity evaluation schema with bilateral MC38 bearing mice were treated with Combo, αOX40, and control. (H) Tumor growth curves and survival plots of (G) ( n = 6–7 mice per group). Data are shown as means ± SD from one of two independent experiments (A–D, F, and H). Statistical significance was determined using log rank test (A–H). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Control

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: The antitumor efficacy of the Combo therapy is contingent upon CD8 + T cells and macrophages (A) UMAP of scRNA-seq data from tumor-infiltrating immune cells in OX40-humanized MC38-bearing mice treated with MPLA, IFN-γ, αOX40, or Combo. Cells are color-coded by annotated cell type. (B) Bubble chart showing the top variable marker genes for identified immune cell types. (C) Pie chart shows the relative abundance of 11 immune cell clusters in control, αOX40, MPLA+IFN-γ, or Combo. (D) Macrophage frequency and absolute count in tumors of MC38-bearing mice after two and three treatment cycles with MPLA, IFN-γ, αOX40, or Combo, analyzed by flow cytometry ( n = 5 mice per group). (E) Schematic of CD8 + T cell depletion assay. (F) Tumor volume and survival were monitored. Kaplan-Meier survival analysis corresponding to the depletion study of CD8 + T cell ( n = 6 mice per group). (G) Schematic of macrophage depletion assay in early and late stage. (H and I) Tumor volume and survival were monitored. Kaplan-Meier survival analysis corresponding to the depletion study in (G) ( n = 6–10 mice per group). Data are shown as means ± SD from one of two independent experiments (D, F, H, and I). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (D). Log rank test was used (F, H, and I) for statistical comparison. n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Marker, Control, Flow Cytometry, Depletion Assay, Comparison

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-high macrophages are significantly associated with Combo treatment efficacy (A) UMAP of macrophage subclusters from scRNA-seq data of MC38-bearing mice treated with control, MPLA+IFN-γ, αOX40, or Combo. Cells are color-coded by annotated subtype. (B) Bubble chart showing the top variable marker genes for identified macrophage subclusters. (C) Pie chart shows the relative abundance of four macrophage subclusters in control, αOX40, MPLA+IFN-γ, or Combo. (D) GO pathway analysis identifying significantly enriched signaling pathways in the Mac_S2 subcluster compared to other macrophage subpopulations. (E) Violin plots showing Nos2 expression levels across macrophage subclusters. (F) Violin plots comparing Nos2 and Cd206 expression levels among different treatment groups. (G) Frequency of M1-like, M2-like, or the ratio of M1/M2-like macrophage cells in tumor tissues from control, αOX40, MPLA+IFN-γ, and Combo groups with two time points, as determined by flow cytometry ( n = 5–10 mice per group). (H) Multiple immunofluorescence signal intensities of NOS2 + F4/80 + and CD206 + F4/80 + cells in the TME of control, αOX40, MPLA+IFN-γ, and Combo groups. Scale bars, 20 μm. Data are shown as means ± SD from one of two independent experiments (G and H). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (G). n.s., not significant; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Control, Marker, Protein-Protein interactions, Expressing, Flow Cytometry, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: NOS2-dependent direct tumor cell killing by macrophages in Combo therapy (A) Schematic of co-culture using CFSE-labeled MC38 cells with tumor- or spleen-derived macrophages, with/without NIL treatment. (B) Flow cytometry quantification of 7-AAD + MC38 cells after 48 h co-culture with tumor-(right) or spleen (left)-derived macrophages ( n = 3 biological replicates). (C) Quantification of 7-AAD + MC38 cells after 48 h in vitro co-culture with or without NIL treatment ( n = 3 biological replicates). (D) Treatment schedule for MC38- or B16-tumor-bearing Nos2 KOor WT mice treated with Combo ( n = 6 mice per group). (E and F) Survival curves of MC38-bearing (E) and B16-bearing mice (F) were analyzed using the log rank test. (G) Flow cytometry analysis of 7-AAD + MC38 cells after 48-h co-culture with CFSE-labeled MC38 cells and BMDMs from Nos2 KO mice ( n = 5 biological replicates). (H) Phagocytosis rate of MC38 cells engulfed by BMDMs was assessed by flow cytometry ( n = 3 biological replicates). (I) Surface expression of CRT on MC38 cells was assessed by flow cytometry after co-culture with MPLA- and IFN-γ-polarized BMDMs in vitro ( n = 3 biological replicates). (J) Analysis of CALR + MC38 cells from MC38-tumor-bearing mice following final treatment with control or Combo, assessed by flow cytometry ( n = 5 biological replicates). Data are shown as means ± SD from one of two independent experiments (B, C, E, F, G, H, I, and J). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons (B, C, and I) or using unpaired Student’s t test (G, H, and J). Log rank tests (E and F) were also used for statistical analysis. n.s., not significant; ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Combo, MPLA, and IFN-γ combined with αOX86 (E and F).

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Co-Culture Assay, Labeling, Derivative Assay, Flow Cytometry, In Vitro, Expressing, Control

Journal: Cell Reports Medicine

Article Title: Immunogenic tumor cell death and T-cell-derived IFN-γ elicit tumoricidal macrophages to potentiate OX40 immunotherapy

doi: 10.1016/j.xcrm.2026.102699

Figure Lengend Snippet: Foxp3 + Treg depletion and macrophage reprogramming are involved in the anti-tumor effect of Combo (A) Flow cytometry analysis of TME. MC38-tumor-bearing mice were treated with control, αOX40, MPLA+IFN-γ, or Combo for two and three doses, and tumors were analyzed by flow cytometry. (B) Frequency and absolute count of CD25 + FOXP3 + cells in tumor tissues from control, αOX40, MPLA+IFN-γ, and Combo groups with two time points, as determined by flow cytometry ( n = 5 mice per group). (C) Treatment schedule for MC38-tumor-bearing Fcer1g KO or FcγRIIb KO mice. Mice were treated with Control, αOX40, MPLA+IFN-γ, and Combo every 3 days for a total of four doses. (D and E) Survival curves of Fcgr1g KO (D) and FcgrIIb KO (E) mice following treatment ( n = 5–6 mice per group) were monitored. (F) Treatment schedule. MC38-tumor-bearing mice were treated with MPLA and IFN-γ in combination with either OX40-mIgG2a or OX40-hIgG1 agonist antibodies (top), and the corresponding survival curves are shown (bottom) ( n = 5–7 mice per group). (G) Schematic of the co-culture experiment involving BMDMs and Tregs at a ratio of 1:4 (BMDM:Treg); Nos2 expression was measured by RT-qPCR. (H) Relative expression of Nos2 following the co-culture ( n = 4 biological replicates). (I) Multiple immunofluorescence (mIF) staining of MC38 tumors from mice treated with control, αOX40, MPLA+IFN-γ, or Combo, showing FOXP3 and NOS2 expression in border or intra-tumoral. Scale bars, 50 μm. (J) Analysis of cell numbers of FOXP3 and NOS2 expression at the border and intra-tumoral. Representative images from five randomly chosen fields were quantified with ImageJ. Data are shown as means ± SD from one of two independent experiments (B, D, E, F, H, and I). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons (B and H). Log rank test was also used (D–F). n.s., not significant; ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Combo, MPLA, and IFN-γ combined with αOX86 (C–E).

Article Snippet: Mouse IFNγ precoated ELISPOT kit , Dakewe , Cat# 221000.

Techniques: Flow Cytometry, Control, Co-Culture Assay, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Coordinated Tuning of Ionizable Lipids and Formulation Redirects mRNA Vaccines Toward Lymphoid-Specific CD4 + T Cell Immunity

doi: 10.64898/2026.04.16.719092

Figure Lengend Snippet: (a) Vaccination scheme. BALB/c mice were immunized intramuscularly with SARS-CoV-2 spike mRNA-loaded LNPs (0.25 mg/kg) following a prime–boost regimen (day 0 and day 21). Blood samples and splenocytes were collected three weeks after the booster dose for antibody and T cell analysis. (b) Antigen-specific IgG levels at three weeks post-boost (n = 5 per group). (c) Neutralizing antibody titers assessed by PRNT₅₀ at three weeks post-boost (n = 3 per group). (d) Antigen-specific T cell responses assessed by IFN-γ ELISpot (n= 2–3 per group). (e) Viral challenge scheme. K18-hACE2 transgenic mice were immunized following the same prime–boost regimen and challenged with SARS-CoV-2 three weeks after the booster dose. (f) Body weight changes following viral challenge (n = 5 per group). (g) Survival curves following viral challenge (n = 5 per group). Statistical significance for (b–d) was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Survival curves were compared using the log-rank (Mantel-Cox) test. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The assay was performed using a mouse IFN-γ ELISpot kit (XEL485, R&D Systems) according to the manufacturer’s instructions.

Techniques: Cell Analysis, Enzyme-linked Immunospot, Transgenic Assay