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: Human Gene Therapy

Article Title: Evaluation of Adeno-Associated Viral Vectors for Liver-Directed Gene Transfer in Dogs

doi: 10.1089/hum.2010.194

Figure Lengend Snippet: Time course of T cell responses to GFP and capsid in peripheral blood mononuclear cells (PBMCs) and lymphocytes isolated from spleen and liver, determined by IFN-γ ELISpot assay. T cells were stimulated with GFP or peptide pools (A, B, C) corresponding to AAV2 and AAV8 capsid, respectively, and the number of IFN-γ-secreting cells in response to antigen stimulation was determined. T cell responses to GFP can be observed in the two animals that received AAV8 on day 27. SFC, spot-forming cells.

Article Snippet: Enzyme-linked immunospot (ELISpot) assays to detect canine interferon (IFN)-γ were performed with the canine IFN-γ ELISpot development module and ELISpot blue color module (R&D Systems, Minneapolis, MN), in accordance with the manufacturer's instructions.

Techniques: Isolation, Enzyme-linked Immunospot

Journal: Human Gene Therapy

Article Title: Evaluation of Adeno-Associated Viral Vectors for Liver-Directed Gene Transfer in Dogs

doi: 10.1089/hum.2010.194

Figure Lengend Snippet: Development of T cells against transgene and capsid in animal S468 determined by IFN-γ ELISpot. Top: Time course of T cell frequencies in PBMCs against GFP and AAV8 capsid (peptide pools 8A, 8B, 8C; control, C). Bottom: Frequencies of GFP and capsid-specific T cells isolated from spleen and liver compared with PBMCs on day 35 after vector administration.

Article Snippet: Enzyme-linked immunospot (ELISpot) assays to detect canine interferon (IFN)-γ were performed with the canine IFN-γ ELISpot development module and ELISpot blue color module (R&D Systems, Minneapolis, MN), in accordance with the manufacturer's instructions.

Techniques: Enzyme-linked Immunospot, Control, Isolation, Plasmid Preparation

Journal: Cell Reports Methods

Article Title: High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay

doi: 10.1016/j.crmeth.2022.100267

Figure Lengend Snippet: Comparison of conventional ELISpot/FluoroSpot assays with plasmon-enhanced FluoroDOT assay relying on plasmonic-fluors as ultrabright biolabels (A and B) Schematic illustration depicting step-by-step method for (A) conventional ELISpot and FluoroSpot assays and (B) plasmon-enhanced FluoroDOT assay. (C–E) Representative whole-well image (scale bar, 1000 μm) and zoomed-in image (scale bar, 100 μm) of PBMCs IFNγ assay. (C) ELISpot, (D) FluoroSPOT, and (E) FluoroDOT. The cells were incubated for 18 h for comparison of all three assays. (F) JAWS II dendritic cells (DCs) were treated with 200 and 2,000 ng/mL lipopolysachharide (LPS), and bright-field images (left panel) and Cy5/DAPI merged images (middle panel) of a single cell secreting TNF-α, visualized using conventional fluor (Cy5), quantum dot 655, and Cy5-plasmonic-fluor. (G) Representative line scans and signal-to-noise ratio (SNR) corresponding to conventional fluor (Cy5), quantum dot 655, and Cy5-plasmonic-fluor. To assess the signal of the secreted cytokine, the line scans for all fluors are placed just outside the boundary of the cell (using the corresponding bright-field image). High-power laser (Lumencor Aura III Light Engine) was used as light source. A standard Cy5 filter was used to image all three labels. See also Figures S1 and .

Article Snippet: Human IFNγ ELISpot development module (R&D systems, catalog number SEL285) was used to perform the FluoroDOT assays.

Techniques: Comparison, Enzyme-linked Immunospot, Incubation

Journal: Cell Reports Methods

Article Title: High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay

doi: 10.1016/j.crmeth.2022.100267

Figure Lengend Snippet: Detection of IFN-γ secretion in a multi-cellular co-culture system (A) Illustration depicting the experiment performed on wild-type (WT) and class II mismatched (MM) bone marrow-derived dendritic cells (BMDCs) infected with Mycobacterium tuberculosis ( Mtb ) and co-cultured with T cells (BMDCs: CD4 + T cells = 1:10). Antigen presentation leads to the activation of green fluorescent protein (GFP)-expressing CD4 + T cells and secretion of IFN-γ. (B and C) IFN-γ secretion by Mtb MHCI-peptide-specific T cells when co-cultured with (B) wild-type (WT) BMDCs infected with Mtb and (C) mismatched (MM) BMDCs infected with Mtb . Epifluorescence microscopy images of the assay using Cy5-plasmonic-fluor as the label. Scale bar: 100 μm. (D) IFN-γ secretion by Mtb MHCI-peptide (P25)-specific T cells when co-cultured with BMDCs for 1, 2, and 3 h. Cy5 (red) epifluorescence microscopy images of the assay using Cy5-plasmonic-fluor. Representative 60× image co-cultures of BMDCs and T cells (DAPI staining nuclei) along with GFP expression (green) triggered in activated CD4 + T cells. White arrows highlight dendritic cells (DCs) (bigger, blue nucleus stained) and T cells (smaller, green cells). Scale bar: 50 μm. (E and F) Quantification of secreted IFN-γ represented as (E) number of particles in each cluster and (F) concentration in pg/mL of IFN-γ protein. Number of particles/clusters were calculated from 35 clusters using twenty 20× images for each treatment condition. ND, not detectable. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by one-way ANOVA and Tukey’s post test. See also Figures S10 and .

Article Snippet: Human IFNγ ELISpot development module (R&D systems, catalog number SEL285) was used to perform the FluoroDOT assays.

Techniques: Co-Culture Assay, Derivative Assay, Infection, Cell Culture, Immunopeptidomics, Activation Assay, Expressing, Epifluorescence Microscopy, Staining, Concentration Assay

Journal: Cell Reports Methods

Article Title: High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay

doi: 10.1016/j.crmeth.2022.100267

Figure Lengend Snippet:

Article Snippet: Human IFNγ ELISpot development module (R&D systems, catalog number SEL285) was used to perform the FluoroDOT assays.

Techniques: Virus, Derivative Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Software, Microscopy, Spectrophotometry, Transmission Assay

Journal: FASEB bioAdvances

Article Title: AJP001, a novel helper T‐cell epitope, induces a humoral immune response with activation of innate immunity when included in a peptide vaccine

doi: 10.1096/fba.2019-00056

Figure Lengend Snippet: Vaccination with AJP001 induces antibody production and T‐cell activation in mice. A. Ang II, a mixture of Ang II and AJP001 or the AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA three times at 2‐week intervals. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). B, C. The AJP001‐Ang II conjugate vaccine (AJP001‐Ang II 100 μg) was administered intracutaneously to 7‐week‐old female BALB/cA (B) or 7‐week‐old male C57BL/6 (C) mice three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum sample collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). D. The AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA mice at a dose of 20, 100, or 500 μg per mouse three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was measured by ELISA. The titers are expressed as the dilution fold of the serum giving half‐maximal absorbance at 450 nm. All data are expressed as the mean ± SD (n = 3). * P < .05, ** P < .01 and *** P < .001 vs the saline group. E, F. Antigen‐specific activation of T cells in AJP001‐Ang II‐immunized mice was evaluated by an ELISpot assay. Splenocytes were isolated from AJP001‐Ang II‐immunized mice and stimulated with Angiotensin II or AJP001 at a concentration of 10 μg/mL. PMA and ionomycin (100 ng/m each) were added to positive control wells, and medium was added as a negative control. The number of IFN‐γ‐ (E) or IL‐4‐producing (F) cells was detected by counting spots using a stereomicroscope. The number of spots was quantified in the duplicate or triplicate wells of each mouse. The data represent the mean ± SD (n = 3). * P < .05 and ** P < .01 vs the saline group

Article Snippet: Mouse IFN‐γ ELISpot Development Module, Mouse IL‐4 ELISpot Development Module and ELISpot Blue Color Module were obtained from R&D Systems, Inc.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Adjuvant, Saline, Enzyme-linked Immunospot, Isolation, Concentration Assay, Positive Control, Negative Control