anti duck cd8 mab  (Bio-Rad)

Journal: Journal of Virology

Article Title: Recombinant duck enteritis virus harboring the hemagglutinin genes of influenza virus rapidly induces specific cellular immunity in ducks

doi: 10.1128/jvi.02014-25

Figure Lengend Snippet: Expansion of T cells induced by rDEV-dH5/H7 and vDEV. Four-week-old ducks were inoculated i.m. with two doses of 10 5 TCID 50 of rDEV-dH5/H7 or vDEV at a 3-week interval. The percentages of ( A ) CD3 + T cells, ( B ) CD3 + CD8 + T cells, and ( C ) CD3 + CD4 + T cells among the PBMCs were analyzed by flow cytometry. Data are presented as means and plotted with connecting lines. Each data point represents one sample value. The red triangles represent the time points of inoculation.

Article Snippet: The antibody cocktail included an FITC-conjugated anti-CD3 mAb (CD3-12; Abcam, ab11089) together with a PE-conjugated anti-duck CD4 mAb (Du CD4-2; Bio-Rad, MCA2478) or a PE-conjugated anti-duck CD8 mAb (Du CD8-1; Bio-Rad, MCA2479), which were labeled with a PE/R-phycoerythrin Conjugation Kit (Abcam, ab102918), respectively.

Techniques: Flow Cytometry

Journal: Journal of Virology

Article Title: Recombinant duck enteritis virus harboring the hemagglutinin genes of influenza virus rapidly induces specific cellular immunity in ducks

doi: 10.1128/jvi.02014-25

Figure Lengend Snippet: Specific CD8 + T-cell responses induced by rDEV-dH5/H7 and vDEV. (A) Timeline of inoculation and sample testing. Arrows indicate inoculation, and red circles indicate bleeds, tests, and post-vaccination time points. ( B ) Frequency of specific CD3 + CD8 + T cells expressing IFN-γ + induced by DEV virion. ( C–F ) Frequency of HA-specific CD3 + CD8 + T cells expressing IFN-γ + . PBMCs were stimulated with single inactivated virus antigens: GZ/S4184 (H5N6), LN/SD007 (H5N1), GX/SD098 (H7N9), or a mixture of antigens of these three influenza virus antigens; CD3 + CD8 + IFN-γ + T cells were then quantified. ( G and H ) Uncorrelated control. CD3 + CD8 + IFN-γ + T cells were quantified after PBMCs were stimulated with WSN (H1N1) or La Sota (NDV). Data are presented as means in histograms. Each data point represents one sample value. Statistical significance: * P < 0.05, ** P <0.01, and *** P < 0.001. P values were determined using a two-tailed unpaired Student’s t test. ( I ) Representative gating strategy for HA-specific CD3 + CD8 + T cells expressing IFN-γ + detected at 31 days post-prime vaccination, following stimulation with the influenza virus antigens mixture. RPMI 1640 and PMA plus ionomycin served as negative and positive controls, respectively.

Article Snippet: The antibody cocktail included an FITC-conjugated anti-CD3 mAb (CD3-12; Abcam, ab11089) together with a PE-conjugated anti-duck CD4 mAb (Du CD4-2; Bio-Rad, MCA2478) or a PE-conjugated anti-duck CD8 mAb (Du CD8-1; Bio-Rad, MCA2479), which were labeled with a PE/R-phycoerythrin Conjugation Kit (Abcam, ab102918), respectively.

Techniques: Expressing, Virus, Control, Two Tailed Test

Journal: Journal of Virology

Article Title: Recombinant duck enteritis virus harboring the hemagglutinin genes of influenza virus rapidly induces specific cellular immunity in ducks

doi: 10.1128/jvi.02014-25

Figure Lengend Snippet: Expansion of T cells induced by rDEV-dH5/H7 and vDEV. Four-week-old ducks were inoculated i.m. with two doses of 10 5 TCID 50 of rDEV-dH5/H7 or vDEV at a 3-week interval. The percentages of ( A ) CD3 + T cells, ( B ) CD3 + CD8 + T cells, and ( C ) CD3 + CD4 + T cells among the PBMCs were analyzed by flow cytometry. Data are presented as means and plotted with connecting lines. Each data point represents one sample value. The red triangles represent the time points of inoculation.

Article Snippet: The PBMCs were plated in 1.5 mL centrifuge tubes (10 6 cells/tube) and fixed with 100 μL of 0.3% paraformaldehyde for 20 min at room temperature and subsequently stained for 30 min at room temperature with either mouse anti-duck CD4 mAb (Du CD4-2; Bio-Rad, MCA2478) or mouse anti-duck CD8 mAb (Du CD8-1; Bio-Rad, MCA2479) diluted in PBS containing 0.05% Tween-20.

Techniques: Flow Cytometry

Journal: Journal of Virology

Article Title: Recombinant duck enteritis virus harboring the hemagglutinin genes of influenza virus rapidly induces specific cellular immunity in ducks

doi: 10.1128/jvi.02014-25

Figure Lengend Snippet: Specific CD8 + T-cell responses induced by rDEV-dH5/H7 and vDEV. (A) Timeline of inoculation and sample testing. Arrows indicate inoculation, and red circles indicate bleeds, tests, and post-vaccination time points. ( B ) Frequency of specific CD3 + CD8 + T cells expressing IFN-γ + induced by DEV virion. ( C–F ) Frequency of HA-specific CD3 + CD8 + T cells expressing IFN-γ + . PBMCs were stimulated with single inactivated virus antigens: GZ/S4184 (H5N6), LN/SD007 (H5N1), GX/SD098 (H7N9), or a mixture of antigens of these three influenza virus antigens; CD3 + CD8 + IFN-γ + T cells were then quantified. ( G and H ) Uncorrelated control. CD3 + CD8 + IFN-γ + T cells were quantified after PBMCs were stimulated with WSN (H1N1) or La Sota (NDV). Data are presented as means in histograms. Each data point represents one sample value. Statistical significance: * P < 0.05, ** P <0.01, and *** P < 0.001. P values were determined using a two-tailed unpaired Student’s t test. ( I ) Representative gating strategy for HA-specific CD3 + CD8 + T cells expressing IFN-γ + detected at 31 days post-prime vaccination, following stimulation with the influenza virus antigens mixture. RPMI 1640 and PMA plus ionomycin served as negative and positive controls, respectively.

Article Snippet: The PBMCs were plated in 1.5 mL centrifuge tubes (10 6 cells/tube) and fixed with 100 μL of 0.3% paraformaldehyde for 20 min at room temperature and subsequently stained for 30 min at room temperature with either mouse anti-duck CD4 mAb (Du CD4-2; Bio-Rad, MCA2478) or mouse anti-duck CD8 mAb (Du CD8-1; Bio-Rad, MCA2479) diluted in PBS containing 0.05% Tween-20.

Techniques: Expressing, Virus, Control, Two Tailed Test

Journal: The Journal of Immunology Author Choice

Article Title: Duck CD8 + T Cell Response to H5N1 Highly Pathogenic Avian Influenza Virus Infection In Vivo and In Vitro

doi: 10.4049/jimmunol.2101147

Figure Lengend Snippet: Monitor of duck survival, H5N1 AIV shedding, HI Ab level, and T lymphocyte percentage postinfection. (A) Survival rate of the mallard ducks (Sheldrake) infected with A/Duck/Guangdong/383/2008 (DK383) virus with the dose of 106 EID50. Both the infected group and the control group consisted of 15 ducks. Curves are significantly different (p < 0.001) by log-rank and Gehan–Breslow–Wilcoxon analyses. (B) Virus (H5N1 AIV) shedding was monitored via detecting the viral load in oropharyngeal and cloacal swabs. Statistical analyses for virus titer in swabs at various time points were performed using one-way ANOVA. (C) HI Ab level was monitored using 1% chicken RBCs. The value >4 (dotted line) was considered HI Ab-positive. One-way ANOVA was used for statistical comparisons. (D and E) The percentage of CD4+ T cells (D) or CD8+ T cells (E) between the control group and infection group at various time points was detected. Cells (2 × 105) from each sample were collected for flow cytometric analysis. A two-way ANOVA was used for statistical comparison. H5N1 virus shedding and H5N1 HI Ab in the control group were all negative at various time points (data not shown). Four ducks of infected and control groups were randomly selected for sampling and detection. *p < 0.05, **p < 0.01, ***p < 0.001. ns, not significant.

Article Snippet: Flow cytometry PBMCs (2 × 10 5 ) were incubated with mouse anti-duck CD4 + mAb (Bio-Rad, Hercules, CA) and mouse anti-duck CD8α + mAb (Abcam, Cambridge, MA), respectively, at 4°C for 30 min. After washing with PBS twice, labeled cells were further incubated with FITC-conjugated mouse IgG (Abbkine, Shanghai, China) in the dark at 4°C for 30 min.

Techniques: Infection, Virus, Control, Comparison, Sampling

Journal: The Journal of Immunology Author Choice

Article Title: Duck CD8 + T Cell Response to H5N1 Highly Pathogenic Avian Influenza Virus Infection In Vivo and In Vitro

doi: 10.4049/jimmunol.2101147

Figure Lengend Snippet: Analysis of the duck CD8+ cell phenotype. (A) Gating strategy for duck CD8+ cells. (B) Analysis of the percentage and phenotype of CD8+ cells in PBMCs of H5N1-infected duck no. 16 and control duck no. 3 at various time points. (C) Contour plot and histogram of CD8low+ and CD8high+ cell populations in PBMCs of three ducks in both the H5N1-infected group and control group at 9 dpi. Cells (2 × 105) from each sample was collected for flow cytometric analysis.

Article Snippet: Flow cytometry PBMCs (2 × 10 5 ) were incubated with mouse anti-duck CD4 + mAb (Bio-Rad, Hercules, CA) and mouse anti-duck CD8α + mAb (Abcam, Cambridge, MA), respectively, at 4°C for 30 min. After washing with PBS twice, labeled cells were further incubated with FITC-conjugated mouse IgG (Abbkine, Shanghai, China) in the dark at 4°C for 30 min.

Techniques: Infection, Control

Journal: The Journal of Immunology Author Choice

Article Title: Duck CD8 + T Cell Response to H5N1 Highly Pathogenic Avian Influenza Virus Infection In Vivo and In Vitro

doi: 10.4049/jimmunol.2101147

Figure Lengend Snippet: WGCNA analysis of duck CD4, CD8, CD8low+, and CD8high+ cell populations in PBMCs at 9 dpi. The CD4+ T or CD8+ T cells in the PBMC pool were collected from three control and three H5N1 AIV-infected ducks, respectively, at 9 dpi (control_CD4, H5_CD4, control_CD8, and H5_CD8), and CD8low+ and CD8high+ cell populations in the PBMC pool from three H5N1 AIV-infected ducks at 9 dpi (H5_CD8low and H5_CD8high) were sorted out for Smart-Seq2. (A) PCA of the six cell populations, each with five replications. The x-axis and y-axis show the principal component (PC)1 and PC2, which explains 42.1 and 31.1% of the total variance. (B) Heatmap of sample expression pattern. Red indicates high-level expression. Green indicates low-level expression. (C) Heatmap of the gene expression pattern of the salmon module. (D) Top 20 KEGG pathways selected for salmon module gene enrichment. (E) Heatmap of the gene expression pattern of the purple module. (F) Top 20 KEGG pathways selected for purple module gene enrichment.

Article Snippet: Flow cytometry PBMCs (2 × 10 5 ) were incubated with mouse anti-duck CD4 + mAb (Bio-Rad, Hercules, CA) and mouse anti-duck CD8α + mAb (Abcam, Cambridge, MA), respectively, at 4°C for 30 min. After washing with PBS twice, labeled cells were further incubated with FITC-conjugated mouse IgG (Abbkine, Shanghai, China) in the dark at 4°C for 30 min.

Techniques: Control, Infection, Expressing, Gene Expression

Journal: The Journal of Immunology Author Choice

Article Title: Duck CD8 + T Cell Response to H5N1 Highly Pathogenic Avian Influenza Virus Infection In Vivo and In Vitro

doi: 10.4049/jimmunol.2101147

Figure Lengend Snippet: Differential gene expression analysis of CD4 or CD8 T cells between the control group and H5N1-infected group. (A) Statistics of differentially expressed genes (DEGs) of CD4 or CD8 T cells after H5N1 AIV infection. (B) Top 20 KEGG pathways selected for DEG enrichment of CD4 T cells. (C) Top 20 KEGG pathways selected for DEG enrichment of CD8 T cells. (D) Heatmap of selected immune-related DEGs from CD8 T cells.

Article Snippet: Flow cytometry PBMCs (2 × 10 5 ) were incubated with mouse anti-duck CD4 + mAb (Bio-Rad, Hercules, CA) and mouse anti-duck CD8α + mAb (Abcam, Cambridge, MA), respectively, at 4°C for 30 min. After washing with PBS twice, labeled cells were further incubated with FITC-conjugated mouse IgG (Abbkine, Shanghai, China) in the dark at 4°C for 30 min.

Techniques: Gene Expression, Control, Infection

Journal: The Journal of Immunology Author Choice

Article Title: Duck CD8 + T Cell Response to H5N1 Highly Pathogenic Avian Influenza Virus Infection In Vivo and In Vitro

doi: 10.4049/jimmunol.2101147

Figure Lengend Snippet: Trend analysis of CD8low+, CD8+, and CD8high+ cell populations of H5N1-infected group at 9 dpi. (A) All DEG expression profiles ordered based on the p values in the order of CD8low+ cells, CD8+ cells (containing CD8low+ and CD8high+ cells), and CD8high+ cells. (B) The DEG expression trends in profile 0. (C) Top 10 KEGG pathways were selected for profile 0 after DEG enrichment. (D) The DEG expression trends in profile 7. (E) Top 10 KEGG pathways were selected for profile 7 after DEG enrichment. (F) Heatmap of selected immune genes. (G) Gene interaction network analysis based on the WGCNA analysis and the STRING database. The gray line indicates the interaction relationship based on the WGCNA analysis. The green line indicates the potential interaction based on the STRING database. Red nodes represent hub genes.

Article Snippet: Flow cytometry PBMCs (2 × 10 5 ) were incubated with mouse anti-duck CD4 + mAb (Bio-Rad, Hercules, CA) and mouse anti-duck CD8α + mAb (Abcam, Cambridge, MA), respectively, at 4°C for 30 min. After washing with PBS twice, labeled cells were further incubated with FITC-conjugated mouse IgG (Abbkine, Shanghai, China) in the dark at 4°C for 30 min.

Techniques: Infection, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: Duck CD8 + T Cell Response to H5N1 Highly Pathogenic Avian Influenza Virus Infection In Vivo and In Vitro

doi: 10.4049/jimmunol.2101147

Figure Lengend Snippet: In vitro culture of H5N1 AIV-specific duck T cells. (A) Screening of optimal H5N1 AIV infection time points with the maximum living cells. The data were collected from three biological samples. The results are presented as means ± SEM, and the unpaired t test was used for statistical comparison. (B) The percentage of NP-expressing cells with various infection dosages. The data were collected from three biological samples. Statistical analysis was performed using one-way ANOVA. (C) Flow cytometric analysis of the NP-positive rate between the infection group and control group. (D) Morphologic observation of memory PBMCs with or without H5N1 AIV stimulation (DK383 strain). Scale bar, 100 μm. Two independent experiments with two duck memory PBMC donors were performed. (E) The proliferation of CFSE-labeled memory PBMCs was detected by CFSE dilution in H5N1-stimulated cells from H5N1-infected ducks after 2 wk of culturing. Red sample indicates the CFSE-labeled memory PBMCs without stimulation. Yellow, green, and black samples represent CFSE-labeled memory PBMCs with H5N1 stimulation after 7, 8, and 14 d of culturing, respectively. (F) The CD4 or CD8 T cell percentage between H5N1-stimulated and unstimulated cells after 14 d of culturing. The unpaired t test was used for statistical comparison. (G) The CD4 or CD8 T cell numbers between H5N1-stimulated and unstimulated cells after 14 d of culturing. T cell percentage or number data were collected from two replicates in two independent experiments. Statistical analysis was performed by an unpaired t test. *p < 0.05, **p < 0.01. ns, not significant.

Article Snippet: Flow cytometry PBMCs (2 × 10 5 ) were incubated with mouse anti-duck CD4 + mAb (Bio-Rad, Hercules, CA) and mouse anti-duck CD8α + mAb (Abcam, Cambridge, MA), respectively, at 4°C for 30 min. After washing with PBS twice, labeled cells were further incubated with FITC-conjugated mouse IgG (Abbkine, Shanghai, China) in the dark at 4°C for 30 min.

Techniques: In Vitro, Infection, Comparison, Expressing, Control, Labeling

Journal: The Journal of Immunology Author Choice

Article Title: Duck CD8 + T Cell Response to H5N1 Highly Pathogenic Avian Influenza Virus Infection In Vivo and In Vitro

doi: 10.4049/jimmunol.2101147

Figure Lengend Snippet: H5N1 AIV-specific duck T cell phenotype and response in vitro. (A) Gating and analysis of the percentage and phenotype of CD4+ T cells between H5N1-stimulated and unstimulated cells after 14 d of culturing. (B) Gating and analysis of the percentage and phenotype of CD8+ T cells between H5N1-stimulated and unstimulated cells after 14 d of culturing. (C) Detection of the H5N1 AIV-specific duck T cell response via qRT-PCR. The data were collected from three replicates in the H5N1-stimulated and unstimulated groups, respectively. The results are presented as means ± SEM, and the paired t test was used for statistical comparison. *p < 0.05, **p < 0.01.

Article Snippet: Flow cytometry PBMCs (2 × 10 5 ) were incubated with mouse anti-duck CD4 + mAb (Bio-Rad, Hercules, CA) and mouse anti-duck CD8α + mAb (Abcam, Cambridge, MA), respectively, at 4°C for 30 min. After washing with PBS twice, labeled cells were further incubated with FITC-conjugated mouse IgG (Abbkine, Shanghai, China) in the dark at 4°C for 30 min.

Techniques: In Vitro, Quantitative RT-PCR, Comparison