Journal: Scientific Reports

Article Title: A canine chimeric monoclonal antibody targeting PD-L1 and its clinical efficacy in canine oral malignant melanoma or undifferentiated sarcoma

doi: 10.1038/s41598-017-09444-2

Figure Lengend Snippet: Enhancement of cytokine production and cell proliferation of dog peripheral blood mononuclear cells by c4G12 treatment. Dog peripheral blood mononuclear cells ( n = 7) were obtained from healthy beagle donors and stimulated by 5 μg/mL staphylococcal enterotoxin B in the presence or absence of 20 μg/mL c4G12. Dog IgG was used as a control antibody. For evaluation of cytokine production, the culture supernatant was harvested on day 3, and concentration of ( a ) IL-2 or ( b ) IFN-γ was measured by ELISA. To evaluate cell proliferation, nucleotide analogue 5-ethynyl-2′-deoxyuridine (EdU) was added to the medium on day 2, and cells were harvested after incubation for another 2 h. The lymphocyte population was gated by forward scatter and side scatter, and the incorporation of EdU in ( c ) CD4+ or ( d ) CD8+ cells was measured by a flow cytometer. Statistical analysis was performed with a Wilcoxon signed rank-sum test.

Article Snippet: The thymidine analogue, EdU, was added to the culture medium at a final concentration of 10 μM on day 2, and cells were harvested after additional incubation for 2 h. Cells were stained with optimal concentrations of anti-canine CD4 antibody (R&D systems) conjugated with PerCP-Cy5.5 using a Lightning-Link Antibody Labeling Kit (Innova Biosciences, Cambridge, UK) and anti-canine CD8 antibody (LifeSpan BioSciences, Seattle, WA, USA) labelled with R-PE using a Zenon Mouse IgG2a Labeling Kit (Thermo Fisher Scientific).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry

Journal: Frontiers in Veterinary Science

Article Title: Canine Transforming Growth Factor-β Receptor 2-Ig: A Potential Candidate Biologic for Melanoma Treatment That Reverses Transforming Growth Factor-β1 Immunosuppression

doi: 10.3389/fvets.2021.656715

Figure Lengend Snippet: Effect of TGF-β1 on Th1 cytokine production and Tregs differentiation in PBMC cultures. (A) IL-2, (B) IFN-γ, and (C) TNF-α concentrations in cell culture supernatant were measured by ELISA. (D) The percentage of CD25 + Foxp3 + cells among CD4 + lymphocytes was measured by flow cytometry. The Wilcoxon signed-rank sum test was used for statistical analysis ( n = 6, * p <0.05).

Article Snippet: Subsequently, 1 μg/mL of anti-canine CD4 mouse monoclonal antibody (R&D systems) and 1 μg/mL phycoerythrin (PE)-conjugated anti-canine CD25 mouse monoclonal antibody (Thermo Fisher Scientific) were added and incubated at 25°C for 30 min.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Frontiers in Veterinary Science

Article Title: Canine Transforming Growth Factor-β Receptor 2-Ig: A Potential Candidate Biologic for Melanoma Treatment That Reverses Transforming Growth Factor-β1 Immunosuppression

doi: 10.3389/fvets.2021.656715

Figure Lengend Snippet: Effect of TGF-βRII-Ig on Th1 cytokine production and Tregs differentiation of in PBMC cultures in the presence of TGF-β1. (A) IL-2, (B) IFN-γ, and (C) TNF-α in cell culture supernatants were measured by ELISA. (D) The percentage of CD25 + Foxp3 + cells among CD4 + lymphocytes was calculated by flow cytometry. The Wilcoxon signed-rank sum test was used for statistical analysis ( n = 6, * p <0.05).

Article Snippet: Subsequently, 1 μg/mL of anti-canine CD4 mouse monoclonal antibody (R&D systems) and 1 μg/mL phycoerythrin (PE)-conjugated anti-canine CD25 mouse monoclonal antibody (Thermo Fisher Scientific) were added and incubated at 25°C for 30 min.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Blood

Article Title: Stable long-term mixed chimerism achieved in a canine model of allogeneic in utero hematopoietic cell transplantation.

doi: 10.1182/blood-2013-11-537571

Figure Lengend Snippet: Figure 1. Development of immune profile in fetal thymus by immunohistochemistry. (A,C,E; 320, H&E) Shows the evolution in tissue architecture, as well as the appearance and expansion of CD4/8 DP cells (B,D,F; 320, CD41 [blue], CD81 [red], CD4/8 DP [purple]). At 33 days (A-B), the thymus is relatively devoid of T cells or their precursors. By 39 days (C-D), DP cells begin to appear and undergo significant expansion by 46 days (E-F), when the tissue architecture takes on a relatively normal postnatal appearance. Scale bars represent 50 mm. Flow cytometry (G) confirms rapid proliferation of DP thymocytes between 39 and 42 days, with even more significant increase by 46 days.

Article Snippet: Primary antibody was applied (mouse anti-canine CD45 [AbD Serotec], goat anti-canine CD4 [R&D Systems, Minneapolis, MN], or mouse anti-canine CD8 [Lifespan, Seattle,WA]) and incubated overnight at 4°C.

Techniques: Immunohistochemistry, Flow Cytometry