mouse ifn γ elispot kit  (R&D Systems)

Journal: Molecular Therapy Oncology

Article Title: Induced pluripotent stem cell-derived dendritic cells potentiate the efficacy of interleukin-12-expressing oncolytic HSV-1

doi: 10.1016/j.omton.2026.201252

Figure Lengend Snippet: Immunological assessments of the combination of T-mfIL12 and immature BMDCs (A) Immunohistochemical analysis of representative MB49 tumors from the indicated treatment groups. Increased intratumoral infiltration of CD8 + lymphocytes was most prominent in the T-mfIL12+BMDC group, followed by the T-01+BMDC group. In both groups, elevated levels of CD4 + lymphocyte infiltration were also observed in the same tumor sections. Other groups, except for the mock group, showed moderate levels of CD4 + infiltration. Scale bars: 100 μm. (B) ELISpot assay for IFN-γ expression revealed the highest number of IFN-γ-producing splenocytes in the T-mfIL12+BMDC group, followed by the T-01+BMDC group. Monotherapies with BMDC, T-01, or T-mfIL12, as well as the mock group, did not result in significant increases. n = 3 per group; all samples were analyzed in triplicate. Error bars represent standard deviation (SD). ∗ p < 0.05, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Cells were plated in triplicates at a density of 1 × 10 5 cells per well in 50 μL of RPMI-1640 medium supplemented with 10% FCS, 2% penicillin-streptomycin, and 50 μM 2-ME, using a MultiScreen 96-well plate (Millipore, USA) pre-coated with anti-mouse IFN-γ monoclonal antibodies from the Mouse IFN-γ ELISpot Kit (AN18; Mabtech AB, cat. #3321-2A).

Techniques: Immunohistochemical staining, Enzyme-linked Immunospot, Expressing, Standard Deviation

Journal: Molecular Therapy Oncology

Article Title: Induced pluripotent stem cell-derived dendritic cells potentiate the efficacy of interleukin-12-expressing oncolytic HSV-1

doi: 10.1016/j.omton.2026.201252

Figure Lengend Snippet: Immunological assessments of the combination of T-mfIL12 and immature iPSDCs (A) Immunohistochemical analysis of representative MB49 tumors from the indicated treatment groups. Increased intratumoral infiltration of CD8 + lymphocytes was observed in both the T-mfIL12+BMDC and T-mfIL12+iPSDC groups. In both groups, CD4 + lymphocyte infiltration was also more prominently observed in the same tumor sections. Other treatment groups, excluding the mock group, exhibited moderate levels of CD4 + T cell infiltration. Scale bars: 100 μm. (B) ELISpot assay showed a high and comparable increase in IFN-γ-producing splenocytes in both the T-mfIL12+BMDC and T-mfIL12+iPSDC groups. n = 3 mice per group; all samples were analyzed in triplicate. Error bars represent standard deviation (SD). ∗∗∗∗ p < 0.0001; NS, not significant.

Article Snippet: Cells were plated in triplicates at a density of 1 × 10 5 cells per well in 50 μL of RPMI-1640 medium supplemented with 10% FCS, 2% penicillin-streptomycin, and 50 μM 2-ME, using a MultiScreen 96-well plate (Millipore, USA) pre-coated with anti-mouse IFN-γ monoclonal antibodies from the Mouse IFN-γ ELISpot Kit (AN18; Mabtech AB, cat. #3321-2A).

Techniques: Immunohistochemical staining, Enzyme-linked Immunospot, Standard Deviation

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: Infection and Immunity

Article Title: Construction and characterization of novel Mycobacterium tuberculosis -derived triple and quadruple knockout vaccines against tuberculosis

doi: 10.1128/iai.00500-25

Figure Lengend Snippet: Rational deletion of genes reduces the intracellular survival and increases the processing of live vaccines through PL fusion and autophagy. ( A ) Deletion of genes in Mtb reduces intracellular viability/growth. BMDMs from naïve C57BL/6 mice were activated with IFN-γ and infected (MOI 1:1) with Mtb strains H37Rv, DKO, TKO-Z, TKO-D, or QKO and incubated. After 1, 4, and 8 days post-infection, cells were washed, lysed, and plated for viable colony counts (CFUs). Data represent the mean ± SD CFUs from triplicate. ( B ) Deletion of genes in Mtb leads to efficient processing through PL fusion. BMDMs from naïve C57BL/6 mice were infected with (a–e) rfpH37Rv, rfpDKO, rfpTKO-D, rfpTKO-Z, and rfpQKO (MOI = 1:1) for 4 h, washed, incubated for 24 h, and stained with primary antibodies to phagosomal maturation marker Rab7, followed by staining with FITC (green) conjugated secondary antibody. Red fluorescent Mtb colocalizing with Rab7 antibodies was scored using a Nikon TiE Fluorescence Microscope and Metaview Deconvolution Software. ( C ) Graph showing percent colocalization of Mtb with Rab7. Percent colocalization was determined by counting 50 macrophages per well, each with 1–3 mycobacteria, and averaging counts from triplicate chambers. One of three similar experiments is shown. ( D ) Deletion of genes in Mtb leads to efficient processing through autophagy. BMDMs from naïve C57BL/6 mice were infected with (a–e) rfpH37Rv, rfpDKO, rfpTKO-D, rfpTKO-Z, and rfpQKO (MOI = 1:1) for 4 h, washed, incubated for 24 h, and stained with primary antibodies to autophagy marker LC3, followed by staining with FITC (green) conjugated secondary antibody. Red fluorescent Mtb colocalizing with LC3 antibodies was scored using a Nikon TiE Fluorescence Microscope and Metaview Deconvolution Software. ( E ) The graph shows the percent colocalization of Mtb with LC3. Percent colocalization was determined by counting 50 macrophages per well, each with 1–3 mycobacteria, and averaging counts from triplicate chambers. One of three similar experiments is shown. Statistical significance was calculated using Student’s t-test. P values below 0.05 ( P < 0.05) are considered significant.

Article Snippet: Mouse IFN-γ ELISpot Plus plates (Catalog #3321-4APT-10, MABTECH Inc., Cincinnati, OH) were washed three times with sterile PBS and then blocked with culture media (RMPI with 10% FBS).

Techniques: Vaccines, Infection, Incubation, Staining, Marker, Fluorescence, Microscopy, Software

Journal: Infection and Immunity

Article Title: Construction and characterization of novel Mycobacterium tuberculosis -derived triple and quadruple knockout vaccines against tuberculosis

doi: 10.1128/iai.00500-25

Figure Lengend Snippet: Rational deletion of genes in Mtb increases the immunogenicity of vaccine strains. ( A ) In vitro antigen presentation. BMDMs from naïve C57BL/6 mice were infected with BCG and Mtb strains (H37Rv, DKO, TKO-D, TKO-Z, and QKO) (MOI = 1:5) for 4 h and cocultured with BB7 T-cell hybridoma specific for Ag85B 241-256 peptide. After 16 h, culture fluids were collected and assayed for IL-2 levels released by the BB7 cells in response to Ag85B peptide. ( B ) Ex vivo immunogenicity to vaccine strains. C57BL/6 mice were vaccinated with BCG, DKO, TKO-D, TKO-Z, and QKO vaccine strains (1 × 10 6 subcutaneously) and the control H37Rv. After 30 days of post-immunization, mice were euthanized, and spleens were isolated. Splenocytes (2.5 × 10 5 /well) were plated and stimulated in vitro for 48 h with Mtb H37Rv whole-cell lysate (20 µg/mL). Supernatants from cultures were collected, and IFN-γ, (a) IL-1β, (b) IL-2, (c) IL-12 (d), and TNF (e) levels were determined by ELISA. ELISpot analysis for IFN-γ-producing splenocytes in vaccinated mice (f). C57BL/6 mice were vaccinated with BCG, DKO, TKO-D, TKO-Z, and QKO vaccine strains and control H37Rv (1 × 10 6 subcutaneously). After 30 days post-immunization, mice were euthanized, and spleens were isolated. Splenocytes (2.5 × 10 5 /well) were plated and stimulated with a combination of Ag85B and CFP-10 peptides in vitro for 48 h. Ag85B/CFP-10 responsive IFN-γ-producing spleen cells were spotted using IFN-γ ELISpot plates following the manufacturer’s protocols. Statistical significance was calculated using Student’s t-test. P values below 0.05 ( P < 0.05) were considered significant.

Article Snippet: Mouse IFN-γ ELISpot Plus plates (Catalog #3321-4APT-10, MABTECH Inc., Cincinnati, OH) were washed three times with sterile PBS and then blocked with culture media (RMPI with 10% FBS).

Techniques: Immunopeptidomics, In Vitro, Infection, Ex Vivo, Control, Isolation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Cell Reports Medicine

Article Title: Preclinical evaluation of an mRNA vaccine developed from the first human isolate of bovine H5N1

doi: 10.1016/j.xcrm.2026.102702

Figure Lengend Snippet: Cellular immune responses elicited by H5 Texas Mut mRNA-LNP (SM102) in mice (A–C) The number of splenocytes secreting IFN-γ (a), TNF-α (b), and IL-2 (c) was detected by ELISpot. (D–F) The percentage of CD4 + T cells secreting IFN-γ (D), TNF-α (E), and IL-2 (F) was quantified by flow cytometry. (G–I) The percentage of CD8 + T cells secreting IFN-γ (G), TNF-α (H), and IL-2 (I) was quantified by flow cytometry. (J) IgG1 and IgG2a subclass Abs against Texas HA were determined, and the ratio of IgG2a/IgG1 was calculated. Data are presented as the mean ± SD, and n = 8 biological replicates for each group (A–J). p values are analyzed with t test (ns p > 0.05, ∗ p < 0.05, ∗∗∗ p < 0.001) (A–J).

Article Snippet: ELISpot Plus: Mouse IFN-γ (HRP) , MabTech , 3321-4HST-2.

Techniques: Enzyme-linked Immunospot, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Preclinical evaluation of an mRNA vaccine developed from the first human isolate of bovine H5N1

doi: 10.1016/j.xcrm.2026.102702

Figure Lengend Snippet: Acute toxicological evaluation of two lipid-delivered vaccines in SD rats (A) Vaccination schedule. SD rats were vaccinated (i.m.) with 50 or 300 μg SM102, or DB-Y delivered mRNA vaccines encoding Mut Texas HA protein or placebo. Blood samples ( n = 8) were collected at 6 h and 2 weeks post-injection (day 14). (B) The body weight growth trend. (C) Local swelling grading of injection site. (D and E) White blood cell (D) and granulocyte (E) counts were analyzed in automatic animal blood cell analyzer. (F and G) IL-6 (F) and IFN-γ (G) cytokines were measured using multiplex immunoassay technology. (H) Binding IgG of serum, reactive to the Texas recombinant secreted HA protein, was measured by ELISA. Data are presented as the mean ± SD; p values are analyzed with t test (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001) (B–H). Titers of placebo group were below detection limit (H).

Article Snippet: ELISpot Plus: Mouse IFN-γ (HRP) , MabTech , 3321-4HST-2.

Techniques: Vaccines, Injection, Multiplex Assay, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay

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