animals 501 madin darby canine kidney mdck cells  (ATCC)

Journal: International Journal of Nanomedicine

Article Title: cGAMP-Loaded M2e Nanovaccine Elicits Cross-Reactive Immunity and Mitigates H6N1 Avian Influenza Infection in Chickens

doi: 10.2147/IJN.S607946

Figure Lengend Snippet: M2e-specific antibody responses and cross-type reactivity induced by the M2e nanoshell vaccine. ( A ) Schematic diagram of the immunization schedule. SPF chickens were divided into three groups (n = 6 per group) and subcutaneously immunized on days 0, 21, and 42 with NS (M2e+cGAMP), M2e formulated with ISA 71 VG (oil-based adjuvant), or left unvaccinated. Blood samples were collected on days 21, 42, 56, 77, and 98 for serological analysis. ( B ) Detection of M2e-specific IgG titers in sera collected at multiple time points after immunization. M2e-specific IgG levels were determined by ELISA, and endpoint titers were defined as the highest serum dilution yielding an OD 4 50 value of 0.2. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test at each time point to compare antibody titers among the three groups. Data are expressed as mean ± SEM. (*p < 0.05). ( C ) Schematic illustration of cross-reactivity ICC testing. Sera from vaccinated chickens were incubated with MDCK cells infected with seven influenza A strains (rgH5N1, H6N1, rgH7N9, H1N1 PR8, H1N2, pdmH1N1, and H3N2) to evaluate antibody recognition of influenza-infected cells expressing M2e. ( D ) Cells infected with different viruses were fixed and incubated with sera collected from chickens immunized subcutaneously with a single dose of NS (M2e+cGAMP), followed by staining with anti-chicken IgG and DAB chromogenic detection. Uninfected MDCK cells served as negative controls. Positive brown staining indicates specific binding of M2e-induced antibodies to influenza-infected cells. (Scale bar = 50 µm).

Article Snippet: The Madin-Darby Canine Kidney (MDCK) cell line (ATCC CCL-34) was used in this study.

Techniques: Adjuvant, Enzyme-linked Immunosorbent Assay, Incubation, Infection, Expressing, Staining, Binding Assay

Journal: International Journal of Nanomedicine

Article Title: cGAMP-Loaded M2e Nanovaccine Elicits Cross-Reactive Immunity and Mitigates H6N1 Avian Influenza Infection in Chickens

doi: 10.2147/IJN.S607946

Figure Lengend Snippet: Isolation of chicken NK cells and ADCC activity induced by vaccinated serum. ( A ) Flow cytometric analysis for isolation of chicken NK cells. Splenocytes isolated from three SPF chickens were pooled and stained with anti-chicken CD3 and CD8α antibodies. NK cells were defined as CD3 − CD8α⁺ and gated based on FSC/SSC profiles. ( B ) Schematic illustration of the in vitro killing assay setup. MDCK cells infected with rgH5N1 (MOI=0.05) were co-incubated with chicken NK cells and vaccinated serum. After 24 h, cell viability was measured using a CCK-8 assay to determine the killing rate. ( C ) Cell-killing rate was assessed using the CCK-8 assay. Data are shown as mean ± SEM. Statistical analysis was performed using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. (***p < 0.001; ****p < 0.0001). Representative cell images of infected MDCK cells ( D ) alone, ( E ) treated with vaccinated chicken serum, ( F ) treated with NK cells, and ( G ) treated with both NK cells and vaccinated chicken serum. Arrows indicate representative regions of reduced cell density and cell detachment, consistent with cytotoxic effects.

Article Snippet: The Madin-Darby Canine Kidney (MDCK) cell line (ATCC CCL-34) was used in this study.

Techniques: Isolation, Activity Assay, Staining, In Vitro, Infection, Incubation, CCK-8 Assay