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: Hypoxia-preconditioned bone marrow–derived mesenchymal stem cells protect neurons from cardiac arrest–induced pyroptosis

doi: 10.4103/NRR.NRR-D-23-01922

Figure Lengend Snippet: HP-BMSCs suppress OGD-induced neuronal pyroptosis to some extent by inhibiting the MAPK and NF-κB pathways. (A) From left to right: BMSCs at passage 3 (P3), osteogenic differentiation BMSCs, and adipogenic differentiation BMSCs. The cells are typically spindle-shaped. Scale bars: 100 μm. (B) Flow cytometric analysis showing the expression of BMSC surface markers. The cells expressed CD29 and CD90 but did not express CD11b and CD45. (C) Immunofluorescence staining of primary cultured neurons for MAP2 (red, Alexa Fluor 594), whereas nuclei were labeled with DAPI (blue). Scale bars: 75 μm. (D) Western blotting analysis was performed to evaluate the expression of NLRP3, GSDMD-N, ASC, cleaved-caspase-1, NF-κB, JNK, and p38 MAPK in primary neurons from the indicated group ( n = 3). The results of parallel test were shown in Additional Figure 2 . (E–K) Quantitative analysis of NLRP3 (E), GSDMD-N (F), ASC (G), cleaved-caspase-1 (H), NF-κB (I), JNK (J), and p38 MAPK (K) protein expression levels as shown in D. Results are expressed as mean ± SD. * P < 0.05, vs . control group; # P < 0.05, vs . OGD group; & P < 0.05, vs . OGD + N-BMSCs group; $ P < 0.05, vs . OGD + HP-BMSCs group (one-way analysis of variance followed by Tukey’s multiple comparisons test). BMSCs: Bone marrow mesenchymal stem cells; HP-BMSCs: hypoxia preconditioned bone marrow mesenchymal stem cells; N-BMSCs: normal-cultured BMSCs; OGD: oxygen-glucose deprivation.

Article Snippet: BMSC-specific surface antigens were detected by flow cytometry (Lin et al., 2020) using CD29-FITC, CD90-PE, CD11b-PE, and CD45-FITC antibodies (BioLegend, San Diego, CA, USA).

Techniques: Expressing, Immunofluorescence, Staining, Cell Culture, Labeling, Western Blot, Control

Journal: Neural Regeneration Research

Article Title: Perfluoropentane-based oxygen-loaded nanodroplets reduce microglial activation through metabolic reprogramming

doi: 10.4103/NRR.NRR-D-23-01299

Figure Lengend Snippet: In vitro biological effects of PFP-OLNDs in BV2 microglia. BV2 microglia were pre-incubated with different concentrations of PFP-OLNDs for 3 hours and then exposed to LPS for 24 hours. (A, B) ROS formation detected by flow cytometry. (C) Representative histograms of CD45, MHC class II and CD86 expression on microglia of different groups. Unstained control (Con) is indicated in the shaded histogram. Blank represents the PBS-treated group. (D) Surface expression (MFI) of CD45, MHC class II and CD86 are shown. (E, F) Western blotting of IL-1β, IL-6, and iNOS. (G) ELISA of IL-1β, IL-6, and TNFα from BV2 cellular supernatant. Data are expressed as mean ± SEM ( n = 3). ** P < 0.01, *** P < 0.001, vs. LPS (one-way analysis of variance followed by Bonferroni post hoc test). DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; ELISA: enzyme-linked immunosorbent assay; IL-1β: interleukin 1 beta; IL-6: interleukin-6; iNOS: inducible nitric oxide synthase; LPS: lipopolysaccharide; MFI: mean fluorescence intensity; MHC: major histocompatibility complex; PBS: phosphate buffered saline; PFP-OLNDs: perfluoropentane-based oxygen-loaded nanodroplets; ROS: reactive oxygen species; TNFα: tumor necrosis factor α.

Article Snippet: Surface staining was then performed using 5 μL of the following antibodies: anti-mouse CD45-PE-Cyanine7 (eBioscience, Cat# 25-0459-42, RRID: AB_1944375), anti-mouse major histocompatibility complex (MHC) class II (IA/I-E)-PE (eBioscience, Cat# 12-5321-82, AB_465928), and anti-mouse CD86 (B7-2)-PE-Cyanine5 (eBioscience, Cat# 15-0862-82, AB_468778) for 20 minutes on ice in the dark.

Techniques: In Vitro, Incubation, Flow Cytometry, Expressing, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Fluorescence, 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: 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