mouse ifn γ elispot kit  (R&D Systems)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative analysis of expression, immunogenicity, and safety profiles between linear and circular RNA vaccine platforms

doi: 10.1016/j.omtn.2026.102954

Figure Lengend Snippet: Cell-mediated immune responses to different RNA platforms Each group includes a negative control (DPBS), unmodified linear mRNA (Lin-WT), modified linear mRNA (Lin-m1Ψ), and unmodified circular RNA (Circ-WT). (A) Immunization schedule schematic diagram for assessing T cell responses. Balb/c mice were intramuscularly immunized twice, 2 weeks apart, with 10 μg of LNP-encapsulated HA-encoding mRNA (Lin-WT, Lin-m1Ψ, or Circ-WT). (B and C) IFN-γ ELISPOT assay showing the number of antigen-specific IFN-γ-secreting splenocytes following immunization with HA-encoding mRNA. (D–F) Flow cytometry analysis of cytokine-producing CD8+ T cells. The frequencies of IFN-γ+, IL-2+, and TNF-α + CD8+ T cells were assessed to evaluate antigen-specific T cell activation. (G–I) Cytokine-producing CD4+ T cells, with frequencies of IFN-γ+, IL-2+, and TNF-α+ CD4+ T cells measured by intracellular cytokine staining. (J) Analysis of double-positive cytokine-expressing CD8+ T cells, indicating polyfunctional T cell responses. (K) Analysis of double-positive cytokine-expressing CD4+ T cells, indicating helper T cell activation. Data represent mean ± SD ( n = 5 per group). Statistical significance was determined by ordinary one-way ANOVA with Tukey’s test or the Kruskal-Wallis test with Dunn’s multiple comparison test, depending on the normality of the data. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: IFN-γ secreting T cells were detected using the ELISpot assay and the mouse IFN-γ ELISpot BASIC kit from Mabtech (Stockholm, Sweden), following the manufacturer’s instructions.

Techniques: Negative Control, Modification, Enzyme-linked Immunospot, Flow Cytometry, Activation Assay, Staining, Expressing, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comparative analysis of expression, immunogenicity, and safety profiles between linear and circular RNA vaccine platforms

doi: 10.1016/j.omtn.2026.102954

Figure Lengend Snippet: Differential innate immune responses induced by different RNA platforms Groups comprise the negative control (DPBS), unmodified linear mRNA (Lin-WT), modified linear mRNA (Lin-m1Ψ), and unmodified circular RNA (Circ-WT). (A) Schematic presentation of in vivo cytokine analysis. Mice were injected intramuscularly with LNP-encapsulated mRNA, and cytokine levels were assessed in serum and lymph nodes at 6- and 24-h post-inoculation. (B–G) In vivo cytokine responses measured in serum and lymph nodes at different time points. Levels of (B-C) IFN-γ, (D) RIG-I, (E) IFN-β, (F) TNF-α, (G) IL-6. mRNA Fold change calculated by the 2 −ΔΔCt method and normalized to GAPDH, with values expressed as fold change relative to the DPBS group. Data represent mean ± SD ( n = 5 per group). Statistical significance was determined by ordinary one-way ANOVA with Tukey’s test or the Kruskal-Wallis test with Dunn’s multiple comparison test, depending on the normality of the data. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.

Article Snippet: IFN-γ secreting T cells were detected using the ELISpot assay and the mouse IFN-γ ELISpot BASIC kit from Mabtech (Stockholm, Sweden), following the manufacturer’s instructions.

Techniques: Negative Control, Modification, In Vivo, Injection, Comparison

Journal: Cell Reports Medicine

Article Title: Spleen-targeted neoantigen mRNA vaccine induces ISG15 + CD8 + T cell-mediated tertiary lymphoid structure formation in hepatocellular carcinoma

doi: 10.1016/j.xcrm.2026.102754

Figure Lengend Snippet: Splenic immune responses mediated by STNvac (A) Schematic illustration showing that STNvac promotes DC activation and antigen-specific T cell induction. (B) Representative immunofluorescence images showing the localization of CD11c + DCs (green) and Cy5-labeled mRNA (red) in the spleen of C57BL/6 mouse 6 h after intravenous administration of LNP-mRNA Cy5 (N/P = 0.5). MZ, marginal zone; WP, white pulp; RP, red pulp. Scale bars, 50 μm. (C and D) Ex vivo IFN-γ ELISpot analysis of STNvac-immunized mice. (C) Representative ELISpot images. (D) Quantification of IFN-γ spot-forming units. (E and F) Flow cytometry analysis of splenic immune cell activation after 48 h of STNvac administration. (E) Representative contour plots. (F) Quantitative analysis. Statistics: one-way ANOVA for (D) and (F). Mean ± SD ( n = 3 biological replicates). Significance levels: ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also and .

Article Snippet: Mouse IFN-γ ELISpot Kit , Mabtech , Cat#3321-4AST-2.

Techniques: Activation Assay, Immunofluorescence, Labeling, Ex Vivo, Enzyme-linked Immunospot, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Spleen-targeted neoantigen mRNA vaccine induces ISG15 + CD8 + T cell-mediated tertiary lymphoid structure formation in hepatocellular carcinoma

doi: 10.1016/j.xcrm.2026.102754

Figure Lengend Snippet: Therapeutic efficacy and tumor microenvironment (TME) alterations induced by STNvac treatment (A) Schematic illustration of the treatment schedule ( n = 7 mice per group). (B) Bioluminescence images showing tumor burden in orthotopic HCC-bearing mice receiving PBS (G1), Neo-mRNA (G2), LNP-scramble mRNA (G3), or STNvac (G4) during the 8-week observation period. (C) Total bioluminescence flux for individual mice corresponding to (B). (D) Survival curves of mice in different treatment groups. (E) Mean body weight of mice monitored throughout the study period, showing no significant loss. (F and G) Flow cytometry analysis of tumor-infiltrating immune cells collected 72 h after the final vaccination (day 10): (F) quantitative analysis ( n = 5 biological replicates) and (G) representative contour plots. (H) Immunofluorescence staining of CD4 + and CD8 + T cells in dissected tumor sections. Scale bars, 50 μm. (I) Cytokine levels (IL-12, IFN-γ, and TNF-α) in tumor lysates measured by ELISA ( n = 5 biological replicates, day 10). Statistics: one-way ANOVA for (F) and (I); log rank (Mantel-Cox) test for (D). Mean ± SD. Significance levels: ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. See also and .

Article Snippet: Mouse IFN-γ ELISpot Kit , Mabtech , Cat#3321-4AST-2.

Techniques: Drug discovery, Flow Cytometry, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Spleen-targeted neoantigen mRNA vaccine induces ISG15 + CD8 + T cell-mediated tertiary lymphoid structure formation in hepatocellular carcinoma

doi: 10.1016/j.xcrm.2026.102754

Figure Lengend Snippet: STNvac-induced enhancement of tumor infiltration, antigen-presenting capacity, and cytotoxic activity of ISG15 + CD8 + T cells (A) UMAP visualization of single-cell transcriptomes from CD45 + immune cells. (B) Relative proportions of major immune cell populations in PBS and STNvac groups. (C) UMAP visualization of T cell clusters from PBS and STNvac groups. (D) Relative proportions of different T cell clusters in PBS and STNvac groups. (E) Violin plots showing expression levels of T cell function markers across T cell clusters. (F) Representative multicolor immunofluorescence images of tumor sections showing co-localization of ISG15 + CD8 + T cells with GZMB and IFN-γ in PBS and STNvac groups. Scale bars, 50 μm. (G) Quantification of ISG15 + CD8 + T cell density co-expressing GZMB and IFN-γ, corresponding to (F). Unpaired two-tailed t test; ∗p < 0.05. Mean ± SD ( n = 3 biological replicates). (H) Comparative GO and KEGG pathway enrichment analyses of ISG15 + CD8 + T cells between PBS and STNvac groups. (I) Kaplan-Meier curves showing 5-year overall survival (OS) and progression-free survival (PFI) for HCC patients in TCGA: LIHC cohort stratified by ISG15 + CD8 + T cell signatures. See also and .

Article Snippet: Mouse IFN-γ ELISpot Kit , Mabtech , Cat#3321-4AST-2.

Techniques: Activity Assay, Single Cell, Expressing, Cell Function Assay, Immunofluorescence, Two Tailed Test

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

Journal: eBioMedicine

Article Title: Nasal RNA-scaffold-protein vaccine protects mice from human H5N1 clade 2.3.4.4b virus lethal infection and safeguards against vaccine-unmatched viruses

doi: 10.1016/j.ebiom.2026.106228

Figure Lengend Snippet: Mutant proteins impacted RSPVac immunisation. (A) Schematic showing the disrupted formation of RSPVac using an RNA-binding-deficient mutant. Female BALF/c mice (n = 6) were nasally immunised with two doses of RSPVac-H5, 14-days apart. The RSPVac was produced with either the WT or mutant protein. Sera, BALF, and BALF-flushed cells were harvested 10 days post-2nd dose and analysed. (B) Fluorescence polarisation of WT and Mutant protein. After mutating major arginine residues, the RNA-binding affinity was reduced by 7-fold. (C–F) ELISA for anti-HA serum IgG, BALF IgA, Serum IgG subtypes, and BALF IgG subtypes. (G) IFNγ ELISpot analysis of BALF-flushed cells, stimulated with the immunogen (WT protein used to generate RSPVac-H5-1194). (A) Schematic of the phase separation mutant of the SARS2-RSPVac. To explore whether phase separation is related to RSPVac nasal immunisation, phase separation mutants (Psmut) were generated and used to generate SARS2-RSPVac. Female BALB/c mice were immunised using the same regimen with WT or Psmut RSPVac. Sera and BALF were harvested for downstream analysis. (B–F) ELISA for anti-spike RBD serum IgG, BALF IgG, BALF IgA, serum IgG1/IgG2a, BALF IgG1/IgG2a. (G) IFNγ ELISpot analysis of BALF-flushed cells, stimulated with the immunogen (protein used to generate WT SARS2-RSPVac). Statistical significance was determined by the Mann–Whitney test. p < 0.05 was considered significant. P-values were shown, and those that were considered statistically not significant were labelled not significant (ns). Figures A were created in BioRender.

Article Snippet: BALF cells were stimulated with recombinant proteins and IFN-secreting cells were detected by ELISpot Flex Mouse IFN-γ (ALP) kit (Mabtech Cat# 3321-2A).

Techniques: Mutagenesis, RNA Binding Assay, Produced, Fluorescence, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Generated, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Nasal RNA-scaffold-protein vaccine protects mice from human H5N1 clade 2.3.4.4b virus lethal infection and safeguards against vaccine-unmatched viruses

doi: 10.1016/j.ebiom.2026.106228

Figure Lengend Snippet: Mouse antibody and T cell responses following RSPVac nasal vaccination. (A) Vaccination regimen. Female BALB/c mice (n = 6–9) were intranasally vaccinated with 2 doses of RSPVac, 14 days apart. Sera, BALF, and BALF cells were harvested at day 10 post-2nd dose for analysis. Control mice received the protein component without RNA (protein-only). Data was shown as box plots with respective colours indicating vaccine given, showing all values. Error bars represented the highest and lowest values of each box. ELISA was used to measure antibody responses for (B) anti-HA serum IgG, (C) anti-HA BALF IgG, (D) anti-HA BALF IgA, (E) anti-NP, and (F) serum anti-HA IgG subtypes for each of the RSPVac, respectively. (G) Mucosal T cell responses measured by IFNγ+ ELISpot using the immunogen (the protein used to generate RSPVac) stimulation of BALF cells. (H) To analyse lung T cells, female BALB/c mice (n = 6) were intranasally vaccinated with 2 doses of RSPVac, 14 days apart. Lungs were harvested at day 7 post-2nd dose. Single cell suspensions were prepared, and the NP peptide pool of H5N1, H1N1, and H7N9 was used to stimulate lung cells. Resident T cell responses were analysed by intracellular staining and FACS analysis. NP-reactive (I) CD4+ and (J) CD8+ T cells were shown, comparing protein-only and RSPVac-vaccinated. Statistical significance was determined by the Mann–Whitney test. p < 0.05 was considered significant. P-values were shown, and those that were considered insignificant were labelled not significant (ns). Figures A and H were created in BioRender.

Article Snippet: BALF cells were stimulated with recombinant proteins and IFN-secreting cells were detected by ELISpot Flex Mouse IFN-γ (ALP) kit (Mabtech Cat# 3321-2A).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Single Cell, Staining, MANN-WHITNEY