Journal: Viruses

Article Title: Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes

doi: 10.3390/v7082818

Figure Lengend Snippet: Analysis of T cell activation in spleen cell cultures of mice immunized with TSVP1-PADRE (left panel) and HaVP1-PADRE (right panel) VLPs. Production of cytokines IL-2 (ng/mL) ( A , B ) and IFN-γ (ng/mL) ( C , D ) by murine spleen cells incubated for 24 h with medium alone, HaVP1-PADRE-4 VLPs, unmodified TSPyV VP1 VLPs, unmodified HaPyV VP1VLPs and ConA was determined by ELISA ( A – D ). The number of IFN-γ-secreting cells treated in the same way was analysed by ELISPOT assay ( E , F ). Mean values and SD obtained from each group of immunized mice (indicated on the x axis of each picture) are given (n = 3). The asterisk (*) indicates statistically significant differences between the VLP- or ConA-treated and untreated spleen cell cultures (*, p < 0.05; **, p < 0.01).

Article Snippet: The cells were seeded into sterile 96-well plates for ELISPOT assays (MAIPSWU10, Merck-Millipore) pre-coated overnight with anti-IFN-γ capture antibody (ELISpot development Module Mouse IFN-γ, R&D, MN, USA) in 100 μL of growth medium containing either ConA (3 μg/mL) or VLPs (HaVP1-PADRE-4, TSPyV VP1 and HaPyV VP1) at a concentration 5 μg/mL.

Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot

Journal: Journal of Clinical Microbiology

Article Title: Human Cytomegalovirus-Specific T-Cell Immune Reconstitution in Preemptively Treated Heart Transplant Recipients Identifies Subjects at Critical Risk for Infection

doi: 10.1128/JCM.06406-11

Figure Lengend Snippet: ELISPOT assay as a predictor of CMV infection. Box-and-whisker plot of CMV-specific IFN-γ ELISPOT levels of HTXs who controlled or experienced CMV viremia within 60 days after ELISPOT determination. The rectangular box connects the upper and lower quartiles and the median. Whiskers connect the upper and lower extremes. The ELISPOT level corresponds to the number of CMV pp65-specific IFN-γ spots/200,000 PBMCs. ELISPOT assay data included those for the test performed from 30 to 360 days posttransplantation. *, P < 0.05.

Article Snippet: PBMCs were resuspended in RPMI 1640 medium supplemented with 10% human type AB serum (Sigma-Aldrich) and seeded at a concentration of 1 × 10 6 cells/ml in 96-well IFN-γ-coated ELISPOT assay plates (Autoimmun Diagnostika).

Techniques: Enzyme-linked Immunospot, Infection, Whisker Assay

Journal: Journal of Clinical Microbiology

Article Title: Human Cytomegalovirus-Specific T-Cell Immune Reconstitution in Preemptively Treated Heart Transplant Recipients Identifies Subjects at Critical Risk for Infection

doi: 10.1128/JCM.06406-11

Figure Lengend Snippet: CMV-specific immunity in 10 pretransplant patients and 48 HTXs. Box-and-whiskers plot of pretransplant (control group), early (<100 days posttransplant), and late (>100 days posttransplant) CMV-specific IFN-γ ELISPOT levels in HTXs. Data are for 19 HTXs with single ELISPOT assay determinations taken <100 or >100 days after transplant and ELISPOT assay and 29 HTXs followed prospectively both before and after 100 days after transplant. Numbers in parentheses display the number of ELISPOT assay determinations done for each group.

Article Snippet: PBMCs were resuspended in RPMI 1640 medium supplemented with 10% human type AB serum (Sigma-Aldrich) and seeded at a concentration of 1 × 10 6 cells/ml in 96-well IFN-γ-coated ELISPOT assay plates (Autoimmun Diagnostika).

Techniques: Enzyme-linked Immunospot

Journal: Journal of Clinical Microbiology

Article Title: Human Cytomegalovirus-Specific T-Cell Immune Reconstitution in Preemptively Treated Heart Transplant Recipients Identifies Subjects at Critical Risk for Infection

doi: 10.1128/JCM.06406-11

Figure Lengend Snippet: Immune reconstitution of 29 HTXs followed prospectively before and after 100 days after transplant. Dotted lines show HTX IFN-γ ELISPOT levels before and after 100 days posttransplant. Arrows indicate the CMV-specific immune response of one HTX who experienced CMV disease. For the other patient experiencing CMV disease, we did not have data from sequential time points.

Article Snippet: PBMCs were resuspended in RPMI 1640 medium supplemented with 10% human type AB serum (Sigma-Aldrich) and seeded at a concentration of 1 × 10 6 cells/ml in 96-well IFN-γ-coated ELISPOT assay plates (Autoimmun Diagnostika).

Techniques: Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Frequency and reactivity of antigen-specific T cells were concurrently measured through the combination of artificial antigen-presenting cell, MACS and ELISPOT

doi: 10.1038/s41598-017-16549-1

Figure Lengend Snippet: Reactivity detection of OVA 257–264 -specific CD8 + T cells by the AAPC-microplate method and traditional ELISPOT assay. After AAPC-bead sorting, the cells retained in each well were further incubated with the AAPC-beads for reactivity evaluation. ( a ) IFN-γ secretion in the OVA 257–264 -specific CD8 + T cell population detected by modified ELISPOT after 24-hr stimulation with the two-signal AAPC-beads in the AAPC-microplate. ( b ) IFN-γ secretion in the spleen cells detected by traditional ELISPOT assay after 24-hr stimulation with OVA 257–264 peptide in a 96-well microplate.

Article Snippet: These kits contain the 96-well transparent polystyrene ELISPOT plates which produced by ANIARA Company (Cat. No: ACT350; West Chester, OH).

Techniques: Enzyme-linked Immunospot, Incubation, Modification

Journal: Scientific Reports

Article Title: Frequency and reactivity of antigen-specific T cells were concurrently measured through the combination of artificial antigen-presenting cell, MACS and ELISPOT

doi: 10.1038/s41598-017-16549-1

Figure Lengend Snippet: Enumeration and reactivity detection of HBc 18–27 /HBs 183–191 -specific CD8 + T cells by AAPC-microplate. PBMCs from all subjects were detected by the AAPC-microplate method and traditional HLA-A2/HBc 18–27 /HBs 183–191 dimers staining plus flow cytometry. ( a ) Magnetic separation of HBc 18–27 /HBs 183–191 -specific CD8 + T cells in the AAPC-microplate. Magnification is 400 × for each image. ( b ) The correlation coefficient between the AAPC-microplate method and flow cytometry as analyzed by two-tailed Pearson correlation. ( c ) The frequencies of HBc 18–27 /HBs 183–191 -specific CD8 + T cells in the PBMCs from all subjects as detected by the AAPC-microplate method and flow cytometry (FACS). ( d ) The reactivity of HBc 18–27 /HBs 183–191 -specific CD8 + T cells (AST) and whole T cells from the HLA-A2-positve patients with chronic Hepatitis B. HBc 18–27 /HBs 183–191 -specific CD8 + T cells retained in micro well after AAPC-bead sorting were further co-incubated with PHA for 24 hrs in the AAPC-microplate. Meanwhile, the PBMC samples from the patients were also co-incubated with PHA for 24 hrs without AAPC-bead sorting. IFN-γ local detection was performed by ELISPOT assay as described and the percentages of IFN-γ-secreting cells in the sorted AST cells and CD3 + T cell populations were calculated. Data are presented as mean ± SD.

Article Snippet: These kits contain the 96-well transparent polystyrene ELISPOT plates which produced by ANIARA Company (Cat. No: ACT350; West Chester, OH).

Techniques: Staining, Flow Cytometry, Two Tailed Test, Incubation, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Frequency and reactivity of antigen-specific T cells were concurrently measured through the combination of artificial antigen-presenting cell, MACS and ELISPOT

doi: 10.1038/s41598-017-16549-1

Figure Lengend Snippet: IFN-γ-positive spots in the AST cell population and whole T cell population from the HLA-A2-positive patient with hepatitis B. ( a ) The IFN-γ-positive spots in the AST cells from ten HLA-A2-positive patients as detected by the AAPC-microplate with PHA stimulation. ( b ) The IFN-γ-positive spots in the T cells from five HLA-A2-positive patients as detected by the traditional ELISPOT assay with PHA stimulation.

Article Snippet: These kits contain the 96-well transparent polystyrene ELISPOT plates which produced by ANIARA Company (Cat. No: ACT350; West Chester, OH).

Techniques: Enzyme-linked Immunospot

Journal: The Journal of Experimental Medicine

Article Title: In vivo switch to IL-10–secreting T regulatory cells in high dose allergen exposure

doi: 10.1084/jem.20080193

Figure Lengend Snippet: Increased allergen-specific Tr1 cells and decreased Th1 and Th2 cells after bee stings. (A) PLA-specific IL-4–, IL-10–, and IFN-γ–secreting CD4 + T cells were purified 12 h after PLA stimulation, and their frequencies were calculated in six beekeepers before the beginning of beekeeping season and after 7 d. They received an average of 13 bee stings. Data are expressed as means + SEM. (B) Frequency of PLA-stimulated cytokine-secreting cells in six beekeepers by ELISPOT assay. (C) Secreted cytokines in PLA-stimulated PBMC cultures were measured by ELISA (IL-4 after 24 h, and IL-13, IFN-γ, and IL-10 after 5 d). (D) PBMCs from beekeepers were stimulated with PLA for 10 d, and intracytoplasmic cytokines were determined 12 h after anti-CD2/-CD3/-CD28 mAb stimulation. The mean percent difference (standard deviations are in parentheses) of eight experiments is shown before and 7 d after multiple bee stings inside each histogram. (E) PLA-specific IL-4–, IL-10–, and IFN-γ–secreting and Der p 1– and Bet v 1–specific IL-10–secreting T cells were purified from beekeepers after multiple bee stings. Their frequency was calculated in CD4 + T cells, and PBMCs were immediately reconstituted by increasing their frequency in cultures by 10 times. Cells were stimulated with PLA, and [ 3 H]thymidine incorporation was determined after 5 d. Three experiments are shown. Data are expressed as means + SEM. *, P < 0.001. i.c., intracutaneous.

Article Snippet: 10 6 PBMCs/ml from six healthy donors were stimulated in 200 μl of medium in 96-well flat-bottom ELISPOT plates for 18 h (Euroclone Ltd.).

Techniques: Purification, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay