mouse ifn γ elispot development module  (R&D Systems)

Journal: bioRxiv

Article Title: ULK1 drives NDP52-mediated selective autophagic degradation of MHC-I to promote immune evasion in HPV-positive head and neck cancer

doi: 10.64898/2026.03.14.711071

Figure Lengend Snippet: Immune cell profiling of tumor tissues from C57BL/6 mice injected with mEERL/sg-Scr or mEERL/sg2- Ulk1 (n=4/group). Isolated single cell suspensions were stained with an antibody cocktail and analyzed by flow cytometry. Percentages of total T cells of CD45 + (A), CD8 + T (B), and CD4 + T cells (C) of total CD3 + cells are shown. T cell profiling of tumor draining lymph nodes (TDLNs) from mice injected with either mEERL/sg-Scr or mEERL/sg- Ulk1 was isolated 21 days post-injection (n=3/group). Single-cell suspensions were stained with an antibody cocktail containing Zombie NIR, anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD44, and anti-CD62L antibodies. Representative bar graphs showing percentages of total T cells (D), CD4 + T (E), and CD8 + T cells (F), effector memory CD8 + T cells (G), and naïve CD8 + T cells (H). P -value was determined by One-way ANOVA. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. Cytolytic activity of isolated CD8 + T cells co-cultured with mEERL cells at a 50:1, 10:1, or 2:1 T cell: tumor cell ratios were measured by LDH assay (I). Effector functions of isolated CD8 + T cells were evaluated via IFNγ production measured by ELISA (J) and ELISpot (K and L). The number of spot-forming cells was counted using NIH ImageJ. All data shown are mean ± SEM and representative of at least 3 biological replicates. P -value was determined using Student’s t- Test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: IFN-γ production was determined using the Mouse IFN-γ ELISpot Development Module (R&D Systems, No. SEL485), visualized using the ELISpot Blue Color Module (Strep-AP and BCIP-BNBT) (R&D Systems, No. SEL002), and imaged using the ImmunoSpot Analyzer (CTL).

Techniques: Injection, Isolation, Single Cell, Staining, Flow Cytometry, Activity Assay, Cell Culture, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Vaccines

Article Title: Immunogenic Comparison of Nucleic Acid-Based Vaccines Administered by Pyro-Drive Jet Injector

doi: 10.3390/vaccines12070757

Figure Lengend Snippet: Comparison of PJI-delivered pDNA and mRNA vaccines on OVA expression and immune response. ( A ) LUC-mRNA (0.2 or 1 µg/20 µL) or CpG-free pDNA encoding LUC (10 and 50 µg/20 µL) were injected intradermally into C57BL/6NJcl mouse backs by PJI ( n = 5 in each group), and luciferase activity in the injected skin tissues was analyzed 3, 6, and 24 h after the injection. Luciferase activity is represented by RLU. ( B ) OVA protein expression 24 h after the intradermal injection of the OVA-encoding CpG-free pDNA (50 µg/20 µL) or mRNA (0.2 or 1 µg/20 µL) into the BALB/c mouse skin ( n = 3 in each group). ( C ) Time course of the experiment. The BALB/c mice were vaccinated twice (prime and boost) intradermally using PJI at a two-week interval and the anti-OVA antibody titer was analyzed at four weeks and IFN-γ ELISpot at five weeks. ( D ) Anti-OVA antibody titer at four weeks after the intradermal prime/boost injections of OVA pDNA (50 µg/20 µL) or mRNA (0.2 or 1 µg/20 µL) into the BALB/c mouse or non-vaccinated control mouse skin by PJI ( n = 5 in each group). ( E ) OVA-specific IFN-γ-secreting splenocytes at five weeks after intradermal prime/boost injections of OVA pDNA (50 µg/20 µL) or mRNA (1 µg/20 µL) into the BALB/c mouse skin by PJI or non-vaccinated mice ( n = 5 in each group). All the results are shown as the mean ± SEM. The p -values were analyzed using the one-way ANOVA test adjusted for multiple testing using Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Mouse IFN-γ ELISpot assay (R&D Systems, Inc., Minneapolis, MN, USA) was performed according to the manufacturer’s instructions.

Techniques: Comparison, Vaccines, Expressing, Injection, Luciferase, Activity Assay, Enzyme-linked Immunospot, Control

Journal: Frontiers in Immunology

Article Title: Recombinant Pichinde viral vector expressing tuberculosis antigens elicits strong T cell responses and protection in mice

doi: 10.3389/fimmu.2023.1127515

Figure Lengend Snippet: Evaluation of TBvac2-induced T cell responses by ELISpot assay. (A) BL6 mice (N=3) were primed with 1x10 5 PFU of rP18tri viral vector (V) alone or TBvac-2 through the IM route, and 21 days later boosted with the same virus at the same dose through the IN route. At 7 d post-boost (dpb), splenocytes were isolated and incubated with medium alone or peptide pools of Ag85B or ESAT-6/EsxA, followed by detection of mouse IFNγ by ELISpot. (B) Representative ELISpot wells. (C) The number of IFNγ-secreting cells per million splenocytes in the vector and TBvac-2-immunized mice after Ag85B or ESAT-6/EsxA peptide stimulation was normalized with medium-alone control and shown as the average with standard deviation. Statistical analysis was conducted with unpaired t-test. **p < 0.01.

Article Snippet: ELISpot was performed using Mouse IFNγ ELISpot Development Module (R&D Systems, SEL485) following the manufacturer’s instruction.

Techniques: Enzyme-linked Immunospot, Plasmid Preparation, Virus, Isolation, Incubation, Control, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Recombinant Pichinde viral vector expressing tuberculosis antigens elicits strong T cell responses and protection in mice

doi: 10.3389/fimmu.2023.1127515

Figure Lengend Snippet: rP18tri-based TB vaccines induced multifunctional CD4 T cells. (A) BL6 mice (N=5) were immunized with rP18tri vector (V), TBvac-2, and TBvac-10 through IN-IN prime-boost strategy. At 7 dpb, PBMCs were collected and incubated in medium only or with pooled Ag85B/ESAT6 antigens, and stained for cell surface markers and cytokines. Representative flow cytometry plots of CD4 T cells (CD44 h CD4 + CD3 + ) positive for dual cytokine expression (IFNγ/IL-2, IFNγ/TNFα, and IL-2/TNFα) after either medium or peptide stimulation were shown for each immunization group (B) . After normalization with the medium control, the average percentages of dual- and triple-positive (IFNγ/IL-2/TNFα) CD4 T cells after peptide stimulation were shown for each group (C) . *p<0.05.

Article Snippet: ELISpot was performed using Mouse IFNγ ELISpot Development Module (R&D Systems, SEL485) following the manufacturer’s instruction.

Techniques: Vaccines, Plasmid Preparation, Incubation, Staining, Flow Cytometry, Expressing, Control

Journal: FEBS Open Bio

Article Title: Enhancement of polyethylene glycol‐cell fusion efficiency by novel application of transient pressure using a jet injector

doi: 10.1002/2211-5463.13557

Figure Lengend Snippet: PJI‐F‐generated DC–tumor cell fusion vaccine elicited antitumor effect. (A) Schematic diagram of DC vaccine mouse tumor model challenge. C57BL/6N mice were subcutaneously injected on the left side with 2 × 10 5 B16‐F10 melanoma cells. DC vaccines were intradermally administered on the opposite side on days 5 and 10. (B) DC–tumor cell fusion vaccine (DC vaccine) was generated by fusing mouse bone marrow‐derived splenocytes with B16‐F10 melanoma cells via nonshaken PEG method (Mix), PEG‐F method (V PEG‐F) or PJI‐F method (V PJI‐F). Naive C57BL/6N mice were immunized with DC vaccine and mouse splenocytes from each group ( n = 4) were harvested 10 days later. Tumor‐specific IFN‐γ‐secreting T cell activation was analyzed using IFN‐γ ELISPOT assay. Nonvaccine‐treated mice (PBS) were used as a control group. * indicates P < 0.05. (C) Tumor size after DC vaccine administration was monitored (all n = 5). Tumor volumes are shown as mean ± SD. P ‐values were analyzed by a two‐tailed Student's t ‐test. * indicates P < 0.05. NS indicates not significant.

Article Snippet: To stimulate splenocytes, B16‐F10 melanoma cells were treated with 15 μg·mL −1 mitomycin C (Nacalai Tesque Inc.) for 45 min. Splenocytes harvested from vaccine‐treated mice and mitomycin C‐treated B16‐F10 melanoma cells were mixed at a ratio of 10 : 1 and co‐cultured for 48 h. Nonadherent splenocytes were collected, and ELISpot assay was performed using the Mouse IFN‐γ Development Module (R&D Systems, Minneapolis, MN, USA) and the ELISpot Blue Color Module (R&D Systems).

Techniques: Generated, Injection, Vaccines, Derivative Assay, Activation Assay, Enzyme-linked Immunospot, Control, Two Tailed Test

Journal: Frontiers in immunology

Article Title: Exploring in vitro expression and immune potency in mice using mRNA encoding the Plasmodium falciparum malaria antigen, CelTOS.

doi: 10.3389/fimmu.2022.1026052

Figure Lengend Snippet: FIGURE 2 Effect of N-glycosylation on in vitro and in vivo responses. (A) Western blot analysis of CHO cells transfected with mRNAs pfceltos ARCA, pfceltos Cap1 and pfceltos ARCA GM. pfceltos mRNA transcripts were codon harmonized (CH) for optimal expression in mice and encoded with the native falciparum celtos signal sequence (Wt-SS). Cell culture supernatants and cell lysates were harvested at 24 hours post-transfection to assess for the effect of N-glycosylation on protein translation. (B) BALB/cJ mice were immunized intramuscularly (IM) two times at a three-week interval with 10µg of pfceltos mRNA encapsulated into LNP1 (10µg LNP1), 10µg pfceltos mRNA with glycosylation site modified (GM) in LNP1 (10µg GM LNP1) or LNP1 alone (n=5 per group). PfCelTOS-specific IgG antibody concentrations (µg/mL) were quantified in sera at pre- immunization, three weeks after the primary dose and two weeks after the final dose by ELISA. Antibody concentrations are reported as the geometric mean and 95% confidence intervals. (C, D) Splenocytes were harvested and IFN-g and IL-4 cytokines were detected by ELISpot assay. The mean number of spot-forming cells (SFC) per splenocytes were reported with standard errors of the mean (SEM). Statistical analysis was performed using Mann-Whitney test (**p<0.01).

Article Snippet: Cytokine response by enzyme linked immunospot (ELISpot) To determine cellular responses against PfCelTOS, mouse IFN-g and IL-4 ELISpot assay (R&D systems SEL485 SEL404, respectively) were performed according to the manufacturer’s instructions.

Techniques: Glycoproteomics, In Vitro, In Vivo, Western Blot, Transfection, Expressing, Sequencing, Cell Culture, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: Frontiers in immunology

Article Title: Exploring in vitro expression and immune potency in mice using mRNA encoding the Plasmodium falciparum malaria antigen, CelTOS.

doi: 10.3389/fimmu.2022.1026052

Figure Lengend Snippet: FIGURE 3 Effect of mRNA dose and nucleoside modification on immune responses. BALB/cJ mice were immunized intramuscularly (IM), two times at three-week intervals, with a low dose (10µg) or a high dose (30µg) of pfceltos mRNA that was N-glycosylation site modified (GM), containing human IgE signal sequence (IgE-SS) (GM IgE-SS), and with nucleoside y-pseudouridine and 5’-methylcytosine substitutions (PU5MC), (GM PU5MC IgE-SS), in LNP (LNP1 or LNP3), (n=5 per group). (A) Antigen-specific IgG antibody concentrations against PfCelTOS were quantified in sera two weeks after the final dose by ELISA. Antibody concentrations are represented as the mean and standard deviation (SD). Statistical analysis was performed using an unpaired t-test (*p<0.05). (B, C) IFN-g and IL-4 cytokines were detected by ELISpot. The mean number of spot-forming cells (SFC) per splenocytes were reported with standard errors of the mean (SEM). Statistical analysis was performed using Mann- Whitney test, (*p<0.05, **p<0.01).

Article Snippet: Cytokine response by enzyme linked immunospot (ELISpot) To determine cellular responses against PfCelTOS, mouse IFN-g and IL-4 ELISpot assay (R&D systems SEL485 SEL404, respectively) were performed according to the manufacturer’s instructions.

Techniques: Glycoproteomics, Sequencing, Enzyme-linked Immunosorbent Assay, Standard Deviation, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: Frontiers in immunology

Article Title: Exploring in vitro expression and immune potency in mice using mRNA encoding the Plasmodium falciparum malaria antigen, CelTOS.

doi: 10.3389/fimmu.2022.1026052

Figure Lengend Snippet: FIGURE 4 A three-dose regime overcomes an “all-or-none” pattern for humoral immune responses. Mice were immunized intramuscularly (IM) thrice at a three-week interval with 10µg of University of Pennsylvania (UPenn) N-glycosylation site modified (GM) and encapsulated in LNP1 (UPenn GM LNP1), or without N-glycosylation modification (UPenn LNP1) that were one-methylpseudouridine (m1Y)-5′-triphosphate modified, and cellulose affinity purified or with TriLink pfceltos mRNA with N-glycosylation site modified (GM) in LNP1 (TriLink GM LNP1) or LNP3 (TriLink GM LNP3) that were y-pseudouridine and 5-methylcytosine modified (n = 15 per group; n = 10 in LNP alone groups). (A) Kinetics of PfCelTOS antibody concentrations measured by ELISA. Antibody concentrations are reported as the geometric mean and 95% confidence intervals. (B) IFN-g cytokine responses were detected by ELISpot, (n=5 per group). The mean number of spot-forming cells (SFC) per splenocytes were reported with standard errors of the mean (SEM). Statistical analysis was performed using Mann-Whitney test, (*p<0.05, **p<0.01).

Article Snippet: Cytokine response by enzyme linked immunospot (ELISpot) To determine cellular responses against PfCelTOS, mouse IFN-g and IL-4 ELISpot assay (R&D systems SEL485 SEL404, respectively) were performed according to the manufacturer’s instructions.

Techniques: Glycoproteomics, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, MANN-WHITNEY