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: PLOS One

Article Title: Rabies vaccination induces a CD4 + T EM and CD4 + CD8 + T EMRA T H 1 phenotype in dogs

doi: 10.1371/journal.pone.0323823

Figure Lengend Snippet: (A) Total IgG titers against RABV-G and RABV-N on the day of a rabies vaccine booster (Day 0) or 7-14 days after vaccine administration. The dashed line was determined by the antibody titer from an unvaccinated puppy (Dog 11, ) and was used as a normalization control for each experiment ( n = 10, paired two-tailed Wilcoxon test; ns, not significant, ** p < 0.01; two independent experiments). (B) Representative IFNγ ELISpot images of mock-, RABV-N-, and RABV-G- stimulated PBMCs performed in triplicate. (C) Quantification of the data represented in (B) ( n = 6; one-way ANOVA and post hoc Tukey’s test; ns, not significant, * p < 0.05, ** p < 0.01; three independent experiments).

Article Snippet: A canine IFNγ ELISpot Development Module (SEL781, R&D Systems) was used per the manufacturer’s protocol with slight modifications.

Techniques: Control, Two Tailed Test, Enzyme-linked Immunospot

Journal: PLOS One

Article Title: Rabies vaccination induces a CD4 + T EM and CD4 + CD8 + T EMRA T H 1 phenotype in dogs

doi: 10.1371/journal.pone.0323823

Figure Lengend Snippet: (A) Representative histogram plots compare the mock control (black outline) to RABV-G-stimulated T cell subsets: CD4 + (top row, blue), DP (middle row, orange), and CD8 + (bottom row, black), with % proliferated (CTFR LO ) shown within the plot. (B) Quantification of (A) (mean ± SEM; n = 6/experiment; two-way ANOVA with multiple comparisons **** p < 0.0001; 6 independent experiments). Data was normalized to controls. (C) Representative histograms of IFNγ (left) and IL-17A (right) expression by CD8 + , CD4 + , or DP T cells stimulated with RABV-G after 5 days. (D) Mean fluorescence intensity (MFI) of IFNγ (left) and IL-17A (right) for CD4 + T cells, DP T cells, and CD8 + T cells (mean ± SEM; n = 6/experiment; two-way ANOVA with multiple comparisons; ns, not significant, * p < 0.05, *** p < 0.001, **** p < 0.0001; 6 independent experiments). MFI was normalized to controls.

Article Snippet: A canine IFNγ ELISpot Development Module (SEL781, R&D Systems) was used per the manufacturer’s protocol with slight modifications.

Techniques: Control, Expressing, Fluorescence

Journal: PLOS One

Article Title: Rabies vaccination induces a CD4 + T EM and CD4 + CD8 + T EMRA T H 1 phenotype in dogs

doi: 10.1371/journal.pone.0323823

Figure Lengend Snippet: (A) Gating strategy and representative contour plots for lineage-defining subsets between CTFR LO and CTFR HI T cells to differentiate T H 1 (T-bet + IFNγ + ), T H 17 (IL-17A + ), T H 1/T H 17 (T-bet + IL-17A + ), pathogenic T H 1/T H 17 (T-bet + IL-17A + IFNγ + ), and Tregs (FOXP3 + ). (B) Quantification of (A) (mean ± SEM, n = 22 samples, paired two-tailed Wilcoxon test; * p < 0.05, *** p < 0.001, **** p < 0.0001; 4 independent experiments).

Article Snippet: A canine IFNγ ELISpot Development Module (SEL781, R&D Systems) was used per the manufacturer’s protocol with slight modifications.

Techniques: Two Tailed Test

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: PLOS Pathogens

Article Title: Dissecting positive selection events and immunological drives during the evolution of adeno-associated virus lineages

doi: 10.1371/journal.ppat.1012260

Figure Lengend Snippet: (A) The sequence of the 15-mer peptide 82, with site 410 being variable among AAV lineages. This T-cell epitope was originally identified in a human subject by the IFN-γ ELISPOT assay . Peptide 82, with glutamine (Q) and threonine (T) at site 410, was hypothesized to represent cognate or variant epitopes with variable affinities recognized by human or mouse MHCII molecules . (B) ELISPOT assay to examine the immunological effects of a mutation at site 410 from Q to T in the AAV2 capsid in a human subject. Approximately 100 human plasma samples were prescreened by ELISA for hu.1 capsid binding, which contains the peptide 82-Q410 epitope with a high prevalence in human populations . PBMCs were then isolated from the positive blood samples to detect IFN-γ secretion by ELISPOT assay after incubation with peptide 82-Q410. Finally, human PBMC samples that were positive according to both ELISA and ELISPOT prescreening were subjected to IFN-γ ELISPOT assays to compare the mutation effects of the peptides 82-Q410 and 82-T410 using a paired t-test. (C), (D) Immunological effects of replacement at site 410 of the AAV2 capsid in BALB/c mice. An MHCII-binding peptide analogous to peptide 82 was predicted by RANKPEP analysis in BALB/c mice. Four BALB/c mice from each group were intramuscularly immunized with Ad-AAV2Cap or PBS. Splenocytes were harvested 9 days after immunization for incubation with the peptides 82-T410 or 82-Q410 before intracellular cytokine staining to detect IFN-γ secretion in CD4+ T cells. Representative flow cytometry images are shown in (C), and an unpaired t-test was performed to compare the reactivity of CD4 + T cells to AAV2 epitopes in (D). (E), (F) Localization of site 410 in the AAV2 structure. A ribbon model of an AAV2 VP3 pentamer is shown from the inside of the capsid (E) and in a side view (F). The 410 sites are represented as sphere models in yellow.

Article Snippet: ELISpot was then used to further identify individuals with active IFN-γ secretion after stimulation with the peptide 82-Q410 via the Human IFN-γ ELISpot Development Module and Color Module (R&D Systems).

Techniques: Sequencing, Enzyme-linked Immunospot, Variant Assay, Mutagenesis, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Binding Assay, Isolation, Incubation, Staining, Flow Cytometry