Journal: Journal of Leukocyte Biology

Article Title: Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice

doi: 10.1093/jleuko/qiae020

Figure Lengend Snippet: Infant mice NK cells do not significantly expand during B. pertussis infection. P7 infant and adult mice were inoculated with B. pertussis or PBS sham inoculum. At 7 dpi, lungs and spleens were harvested for flow cytometry. Top, the numbers of (A) NK cells, (B) NKT-like cells, or (C) T cells are based on the total number of Live CD45 cells in the lungs. Bottom, the numbers of (D) NK cells, (E) NKT-like cells, or (F) T cells are based on the total number of live lymphocytes in the spleen. Symbols represent individual mice (n = 3–7 mice/group), and bars indicate mean ± SEM. Statistical significance was analyzed using Ordinary One-way ANOVA with Tukey’s multiple comparisons test. *p≤0.05, ***p≤0.001, ****p≤0.0001.

Article Snippet: To confirm NK cell purity, 250,000 cells from either unfractionated splenocytes or NK cell enrichment were stained with viability dye 7-AAD (Biolegend), PE-CD45.2 (clone A2,), FITC-, PerCP-cy5.5-, or PE-NK-1.1 (clone PK 136), APC-eF780-CD3e (clone 145–2C11) and PE-Cy7-CD69 and purity was confirmed by flow cytometry.

Techniques: Infection, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice

doi: 10.1093/jleuko/qiae020

Figure Lengend Snippet: Infant mice NK cells produce less IFN-γ than adult NK cells during B. pertussis infection and have an immature phenotype. (A) Schematic outline of the experimental design. P7 infant and adult GREAT (IFN-γ-YFP reporter) mice were inoculated with B. pertussis or PBS, and lungs were harvested for flow cytometry and other assays at 7 dpi. (B) Flow cytometry analysis showing the percentage of IFN-γ-expressing cells among live CD45+ NK, NKT-like, CD4+, and CD8+ T cells in adult mice (upper panels) and infant mice (lower panels). Plots are from single mice and are representative of 4–5 mice per group. (C) Graphed data corresponding to results in (B) from all mice (n = 4–5 mice per group). (D) Histogram showing expression of CD27 on lung NK cells from B. pertussis-infected infant and adult mice. (E) Mean fluorescence intensity of CD27 expression on lung NK cells from B. pertussis-infected infant and adult mice. (F) Production of lung IL-12p70 in B. pertussis-infected and PBS sham-inoculated infant and adult mice. (C, E, and F) Symbols represent individual mice (n = 4–5 mice/group), and bars indicate mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, Ordinary One-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: To confirm NK cell purity, 250,000 cells from either unfractionated splenocytes or NK cell enrichment were stained with viability dye 7-AAD (Biolegend), PE-CD45.2 (clone A2,), FITC-, PerCP-cy5.5-, or PE-NK-1.1 (clone PK 136), APC-eF780-CD3e (clone 145–2C11) and PE-Cy7-CD69 and purity was confirmed by flow cytometry.

Techniques: Infection, Flow Cytometry, Expressing, Fluorescence

Journal: Journal of Leukocyte Biology

Article Title: Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice

doi: 10.1093/jleuko/qiae020

Figure Lengend Snippet: Adoptive transfer of NK cells from adult mice to infant mice reduces bacterial dissemination during B. pertussis infection. (A) Schematic outline of the experimental design. Purified splenic NK cells or whole splenocytes from adult CD45.2 mice (or PBS control) were adoptively transferred to infant CD45.1 mice at P6. At P7, mice were inoculated with B. pertussis and euthanized at 7 dpi, and tissue was harvested for flow cytometry and bacterial CFU assessment. (B) Flow cytometry analysis of the percentage of donor CD45.2+ cells in the lungs or spleen of infant mice receiving no cells, NK cells, or whole splenocytes from adult mice. (C) Number of donor CD45.2+ cells in the lungs or spleen of infant mice receiving no cells, NK cells, or whole splenocytes from adult mice (n = 3–5 per group). (D, E) Bacterial burden in the lungs (D) and blood (E) of B. pertussis-infected infant mice receiving no cells, NK cells, or whole splenocytes from adult mice at 7 dpi (n = 7–14 per group). (C, D, and E) Symbols represent individual mice, and bars indicate mean ± SEM. *p≤0.05, **p≤0.01, ***p≤0.001, Ordinary One-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: To confirm NK cell purity, 250,000 cells from either unfractionated splenocytes or NK cell enrichment were stained with viability dye 7-AAD (Biolegend), PE-CD45.2 (clone A2,), FITC-, PerCP-cy5.5-, or PE-NK-1.1 (clone PK 136), APC-eF780-CD3e (clone 145–2C11) and PE-Cy7-CD69 and purity was confirmed by flow cytometry.

Techniques: Adoptive Transfer Assay, Infection, Purification, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice

doi: 10.1093/jleuko/qiae020

Figure Lengend Snippet: Infant mice NK cells do not significantly expand during B. pertussis infection. P7 infant and adult mice were inoculated with B. pertussis or PBS sham inoculum. At 7 dpi, lungs and spleens were harvested for flow cytometry. Top, the numbers of (A) NK cells, (B) NKT-like cells, or (C) T cells are based on the total number of Live CD45 cells in the lungs. Bottom, the numbers of (D) NK cells, (E) NKT-like cells, or (F) T cells are based on the total number of live lymphocytes in the spleen. Symbols represent individual mice (n = 3–7 mice/group), and bars indicate mean ± SEM. Statistical significance was analyzed using Ordinary One-way ANOVA with Tukey’s multiple comparisons test. *p≤0.05, ***p≤0.001, ****p≤0.0001.

Article Snippet: GREAT (B6.129S4-Ifngtm3.1Lky/J) RRID: IMSR_JAX:017581 [ 12 ] and CD45.1 (B6.SJL-PtprcaPepcb/BoyJ) RRID: IMSR_JAX:002014 mice were purchased from Jackson Labs. All studies were performed on preweaning animals aged postnatal day 7 (P7) to P21 or adult mice 6–8 weeks.

Techniques: Infection, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice

doi: 10.1093/jleuko/qiae020

Figure Lengend Snippet: Infant mice NK cells produce less IFN-γ than adult NK cells during B. pertussis infection and have an immature phenotype. (A) Schematic outline of the experimental design. P7 infant and adult GREAT (IFN-γ-YFP reporter) mice were inoculated with B. pertussis or PBS, and lungs were harvested for flow cytometry and other assays at 7 dpi. (B) Flow cytometry analysis showing the percentage of IFN-γ-expressing cells among live CD45+ NK, NKT-like, CD4+, and CD8+ T cells in adult mice (upper panels) and infant mice (lower panels). Plots are from single mice and are representative of 4–5 mice per group. (C) Graphed data corresponding to results in (B) from all mice (n = 4–5 mice per group). (D) Histogram showing expression of CD27 on lung NK cells from B. pertussis-infected infant and adult mice. (E) Mean fluorescence intensity of CD27 expression on lung NK cells from B. pertussis-infected infant and adult mice. (F) Production of lung IL-12p70 in B. pertussis-infected and PBS sham-inoculated infant and adult mice. (C, E, and F) Symbols represent individual mice (n = 4–5 mice/group), and bars indicate mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001, Ordinary One-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: GREAT (B6.129S4-Ifngtm3.1Lky/J) RRID: IMSR_JAX:017581 [ 12 ] and CD45.1 (B6.SJL-PtprcaPepcb/BoyJ) RRID: IMSR_JAX:002014 mice were purchased from Jackson Labs. All studies were performed on preweaning animals aged postnatal day 7 (P7) to P21 or adult mice 6–8 weeks.

Techniques: Infection, Flow Cytometry, Expressing, Fluorescence

Journal: Journal of Leukocyte Biology

Article Title: Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice

doi: 10.1093/jleuko/qiae020

Figure Lengend Snippet: Adoptive transfer of NK cells from adult mice to infant mice reduces bacterial dissemination during B. pertussis infection. (A) Schematic outline of the experimental design. Purified splenic NK cells or whole splenocytes from adult CD45.2 mice (or PBS control) were adoptively transferred to infant CD45.1 mice at P6. At P7, mice were inoculated with B. pertussis and euthanized at 7 dpi, and tissue was harvested for flow cytometry and bacterial CFU assessment. (B) Flow cytometry analysis of the percentage of donor CD45.2+ cells in the lungs or spleen of infant mice receiving no cells, NK cells, or whole splenocytes from adult mice. (C) Number of donor CD45.2+ cells in the lungs or spleen of infant mice receiving no cells, NK cells, or whole splenocytes from adult mice (n = 3–5 per group). (D, E) Bacterial burden in the lungs (D) and blood (E) of B. pertussis-infected infant mice receiving no cells, NK cells, or whole splenocytes from adult mice at 7 dpi (n = 7–14 per group). (C, D, and E) Symbols represent individual mice, and bars indicate mean ± SEM. *p≤0.05, **p≤0.01, ***p≤0.001, Ordinary One-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: GREAT (B6.129S4-Ifngtm3.1Lky/J) RRID: IMSR_JAX:017581 [ 12 ] and CD45.1 (B6.SJL-PtprcaPepcb/BoyJ) RRID: IMSR_JAX:002014 mice were purchased from Jackson Labs. All studies were performed on preweaning animals aged postnatal day 7 (P7) to P21 or adult mice 6–8 weeks.

Techniques: Adoptive Transfer Assay, Infection, Purification, Flow Cytometry

Journal: Neural Regeneration Research

Article Title: Human-induced pluripotent stem cell–derived neural stem cell exosomes improve blood–brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis

doi: 10.4103/NRR.NRR-D-23-01889

Figure Lengend Snippet: hiPSC–NSC–Exos suppress leukocyte infiltration on day 3 post-ICH. (A) Flow cytometry analysis of neutrophils (CD45 high CD11b + Ly6G + ), macrophages (CD45 high CD11b + F4/80 + ), CD4 + T cells (CD45 high CD3 + CD4 + ), CD8 + T cells (CD45 high CD3 + CD8 + ), NK cells (CD45 high CD3 – NK1.1 + ), and B cells (CD45 high CD3 – CD19 + ) that had infiltrated the brain tissue. (B) Quantification of leukocyte subsets that had infiltrated the brain tissue ( n = 7). (C) Microglial cell counts ( n = 7). (D) Flow cytometry analysis of neutrophils (CD11b + Ly6G + ), macrophages (CD11b + F4/80 + ), CD4 + T cells (CD3 + CD4 + ), CD8 + T cells (CD3 + CD8 + ), NK cells (CD3 – NK1.1 + ), and B cells (CD3 – CD19 + ) that had infiltrated the spleen. (E) Quantification of immune cell subsets that had infiltrated the mouse spleen ( n = 7). Data are presented as mean ± SEM. ** P < 0.01, *** P < 0.001 (Student’s t -test). All statistical values are presented in . Exo: Exosomes; hiPSC: human induced pluripotent stem cell; ICH: intracerebral hemorrhage; NK: natural killer; ns: not significant; NSC: neural stem cell; PBS: phosphate-buffered saline.

Article Snippet: The CD45 antibody was purchased from Invitrogen (Carlsbad, CA, USA), and all other antibodies were purchased from BioLegend (San Diego, CA, USA).

Techniques: Flow Cytometry, Saline

Journal: Neural Regeneration Research

Article Title: Human-induced pluripotent stem cell–derived neural stem cell exosomes improve blood–brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis

doi: 10.4103/NRR.NRR-D-23-01889

Figure Lengend Snippet: Summary of statistical effect sizes in Figure 4

Article Snippet: The CD45 antibody was purchased from Invitrogen (Carlsbad, CA, USA), and all other antibodies were purchased from BioLegend (San Diego, CA, USA).

Techniques: Control