Journal: PLoS ONE

Article Title: Neisseria gonorrhoeae co-infection exacerbates vaginal HIV shedding without affecting systemic viral loads in human CD34 + engrafted mice

doi: 10.1371/journal.pone.0191672

Figure Lengend Snippet: A) Schematic of experimental infection procedure following 18-week engraftment period for CD34 + HSC-transplanted NSG mice, including hormone delivery and antibiotic schedule, as described in Materials and Methods. HIV titres (RNA copies/mL) following i.p. infection with TCID 10 000 HIV-1 BaL in B) plasma and E) vaginal lavage (Open circle = HIV only). HIV titres following vaginal (vag) and transcervical (t.c.) inoculation with N . gonorrhoeae (red circle) or PBS (black circle) in plasma (C,D) and vaginal lavages (F,G). Limit of detection ≤40 copies HIV RNA per 20 μL of plasma or 30 μL of vaginal lavage; not detectable (ND). CD4 + T helper levels in blood (H-J) as measured by flow cytometry (gated on live hCD45 + cells) and analyzed for paired changes using Wilcoxon rank-test with * denoting p≤0.05, no significance (NS). Each replicate denotes samples from one mouse and error bars denote the standard error of the mean. (K) Categorical variable analysis of whether or not N . gonorrhoeae (Ngo) infection resulted in vaginal HIV shedding with data from F and G, * denoting p≤0.05 as determined by Pearson’s chi-square test (two-tailed).

Article Snippet: Antibodies used included anti-mouse CD45 (clone 30-F11) conjugated to PerCP-Cy5.5 (eBiosciences), anti-human CD45 (clone 2D1) conjugated to APC-Cy7 (BD), anti-human CD33 (clone WM53) conjugated to Alexa Fluor 700 (BD), anti-human CD3 (clone UCHT1) conjugated to PE-Cy7 (eBiosciences), anti-human CD4 (RPA-T4) conjugated to APC (BD), anti-human CD8β (clone 2ST8.5H7) conjugated to ECD (Beckman Coulter), anti-human TCRαβ (clone IP26) conjugated to FITC (eBioscience), anti-human CD19 (clone HIB19) conjugated to PE (eBioscience), anti-human CCR5 (clone 2D7/CCR5) conjugated to Brilliant Violet 421 (BD), and live/dead fixable aqua (Molecular Probes).

Techniques: Infection, Flow Cytometry, Two Tailed Test

Journal: PLoS ONE

Article Title: Xenogeneic Graft-versus-Host-Disease in NOD- scid IL-2Rγ null Mice Display a T-Effector Memory Phenotype

doi: 10.1371/journal.pone.0044219

Figure Lengend Snippet: Peripheral blood was collected from Hu-PBMC mice bi-weekly and stained with anti-human specific antibodies for human (A) CD45, (B) CD3, (C) CD20, (D) CD25, and (E) CD45RO and quantified by flow cytometry. 2-way ANOVA test was performed. Each data point represents the average ( n = 4 NSG and BRG mice respectively, 2 PBMC donors) ±SEM values.

Article Snippet: The following antibodies were used in dilutions according to manufacturers instructions: FITC conjugated anti-human CD62L (Invitrogen), PE conjugated anti-human CD27 (eBioscience), PE conjugated anti-human CD25 (BD), PE conjugated anti-human CLA (Miltenyi), PE-Cy5.5 conjugated anti-human CD3 (Invitrogen), PE-Cy5.5 conjugated anti-human CD20 (Invitrogen), PE-Cy7 conjugated anti-mouse CD45 (eBioscience), PE-TR conjugated anti-human CD4 (Invitrogen), APC conjugated anti-human CD8 (Invitrogen), APC-Cy7 conjugated anti-human CD45 (eBioscience), and Pacific Blue conjugated anti-human CD45RO (BD).

Techniques: Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Xenogeneic Graft-versus-Host-Disease in NOD- scid IL-2Rγ null Mice Display a T-Effector Memory Phenotype

doi: 10.1371/journal.pone.0044219

Figure Lengend Snippet: Cells were harvested from the spleens of Hu-PBMC NSG and BRG mice at weekly intervals following transfer and stained with anti-human specific antibodies for mouse CD45 and for human CD45, CD3, CD4, and CD20. Samples were analysed by flow cytometry to calculate the % of human (A) CD45 + , (C) CD3 + and (G) CD20 + , the absolute accumulation of human (B) CD45 + , (D) CD3 + , (E) CD8 + , (F) CD4 + and (H) CD20 + cells. 2-way ANOVA test was performed. Data are compiled from 2 independent experiments (NSG n = 4–5 per time point and BRG n = 4 day 7–21 or n = 2 day 28, 2 PBMC donors).

Article Snippet: The following antibodies were used in dilutions according to manufacturers instructions: FITC conjugated anti-human CD62L (Invitrogen), PE conjugated anti-human CD27 (eBioscience), PE conjugated anti-human CD25 (BD), PE conjugated anti-human CLA (Miltenyi), PE-Cy5.5 conjugated anti-human CD3 (Invitrogen), PE-Cy5.5 conjugated anti-human CD20 (Invitrogen), PE-Cy7 conjugated anti-mouse CD45 (eBioscience), PE-TR conjugated anti-human CD4 (Invitrogen), APC conjugated anti-human CD8 (Invitrogen), APC-Cy7 conjugated anti-human CD45 (eBioscience), and Pacific Blue conjugated anti-human CD45RO (BD).

Techniques: Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Xenogeneic Graft-versus-Host-Disease in NOD- scid IL-2Rγ null Mice Display a T-Effector Memory Phenotype

doi: 10.1371/journal.pone.0044219

Figure Lengend Snippet: Peripheral blood, spleen, lymph nodes, and bone marrow were harvested at GvHD development in Hu-PBMC NSG and BRG mice and stained with anti-human specific antibodies for human (A) CD45, (B) CD3, (C) CD4, (D) CD25, (E) CD45RO, and (F) CD20 and quantified by flow cytometry. Tukey box-and-whisker graphs are shown. Unpaired t test was performed. Data are compiled from 3 independent experiments ( n = 4 NSG and BRG mice respectively, 2 PBMC donors).

Article Snippet: The following antibodies were used in dilutions according to manufacturers instructions: FITC conjugated anti-human CD62L (Invitrogen), PE conjugated anti-human CD27 (eBioscience), PE conjugated anti-human CD25 (BD), PE conjugated anti-human CLA (Miltenyi), PE-Cy5.5 conjugated anti-human CD3 (Invitrogen), PE-Cy5.5 conjugated anti-human CD20 (Invitrogen), PE-Cy7 conjugated anti-mouse CD45 (eBioscience), PE-TR conjugated anti-human CD4 (Invitrogen), APC conjugated anti-human CD8 (Invitrogen), APC-Cy7 conjugated anti-human CD45 (eBioscience), and Pacific Blue conjugated anti-human CD45RO (BD).

Techniques: Staining, Flow Cytometry, Whisker Assay

Journal: PLoS ONE

Article Title: Xenogeneic Graft-versus-Host-Disease in NOD- scid IL-2Rγ null Mice Display a T-Effector Memory Phenotype

doi: 10.1371/journal.pone.0044219

Figure Lengend Snippet: 10 7 freshly isolated PBMCs were injected intravenously via the tail vein into adult 6–12 week old NSG immunodeficient mice 24 hours post sub lethal irradiation (2.4 Gys). (A) Survival of irradiated Hu-PBMC NSG mice (MST = 14 days, n = 7, 2 PBMC donors). (B) The average weight is shown as a percentage of starting weight. Peripheral blood, spleen and bone marrow were harvested from irradiated Hu-PBMC NSG mice at the indicated time points and analysed for (C) human CD45 engraftment and (D) CD4:CD8 ratio. Black bar represents human PBMC phenotype pre-injection. Data represent the mean ±SEM and are compiled from 2 independent experiments (n = 7). (A) Log-rank Mantel-Cox Test, (B, D) unpaired t test, and (C) 2-way ANOVA, spleen vs blood (*,**) and spleen vs bone marrow (##).

Article Snippet: The following antibodies were used in dilutions according to manufacturers instructions: FITC conjugated anti-human CD62L (Invitrogen), PE conjugated anti-human CD27 (eBioscience), PE conjugated anti-human CD25 (BD), PE conjugated anti-human CLA (Miltenyi), PE-Cy5.5 conjugated anti-human CD3 (Invitrogen), PE-Cy5.5 conjugated anti-human CD20 (Invitrogen), PE-Cy7 conjugated anti-mouse CD45 (eBioscience), PE-TR conjugated anti-human CD4 (Invitrogen), APC conjugated anti-human CD8 (Invitrogen), APC-Cy7 conjugated anti-human CD45 (eBioscience), and Pacific Blue conjugated anti-human CD45RO (BD).

Techniques: Isolation, Injection, Irradiation

Journal: The Journal of Clinical Investigation

Article Title: T cells establish and maintain CNS viral infection in HIV-infected humanized mice

doi: 10.1172/JCI98968

Figure Lengend Snippet: (A) Flow cytometric analysis revealed the presence of murine hematopoietic cells (mCD45+) in the brains of BALB/c mice. Murine myeloid cells (mCD11b+), B cells (mCD19+), and T cells (mCD3ε+), including CD4+ and CD8+ T cell subsets, were present. (B) Representative flow cytometric plots from 2 of the BLT mice in Figure 2A demonstrating the presence of human hematopoietic cells (hCD45+), myeloid cells (hCD33+), B cells (hCD19+), and T cells (hCD3+), including CD4+ and CD8+ T cell subsets. (C) Phenotypic characterization of the human macrophages in the brains of BLT mice showed the presence of classical (CD14+CD16–), intermediate (CD14+CD16+), and nonclassical (CD14dimCD16+) macrophages (gating for hCD45+hCD11b+CD33+ cells).

Article Snippet: The antibody panel used to analyze cells isolated from humanized mice included antibodies directed against hCD45 (APC, BD, catalog 555485 or APC-Cy7, BD, catalog 557833); hCD3 (FITC, BD, catalog 555339); hCD4 (APC-H7, BD, catalog 560158); hCD8 (PerCP, BD, catalog 347314); hCD19 (PE-Cy7, BD, catalog 557835); hCD11b (PE, BD, catalog 555388 or APC, catalog 340937); hCD14 (FITC, BD, catalog 555397); hCD16 (PE-Cy7, BD, catalog 557744); and/or hCD33 (PE, BD, catalog 340679).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: T cells establish and maintain CNS viral infection in HIV-infected humanized mice

doi: 10.1172/JCI98968

Figure Lengend Snippet: (A) Total numbers of human hematopoietic cells, T cells, CD4+ T cells, CD8+ T cells, myeloid cells, and B cells in the brains of BLT mice (n = 104). Brains were harvested from BLT mice 5 to 41 week after humanization surgery, and flow cytometry was performed to determine the numbers of human immune cells present. Horizontal lines in A indicate the mean ± SEM. Scatter plots depict the total numbers of human (B) hematopoietic cells (n = 104), (C) T cells (n = 97), (D) CD4+ T cells (n = 97), (E) CD8+ T cells (n = 97), (F) myeloid cells (n = 97), (G) B cells (n = 94). (H) Scatter plot shows the CD4+/CD8+ ratio (n = 97) in the brain and the post-humanization surgery time points at which analysis was performed. (I) Scatter plot depicts the absolute number of human CD45+ cells in the brain and the percentage of human CD45+ cells in the peripheral blood (PB) of BLT mice (n = 103) at necropsy. A Spearman’s rank correlation test was used to analyze the data in B–I, and P values are indicated on the individual graphs.

Article Snippet: The antibody panel used to analyze cells isolated from humanized mice included antibodies directed against hCD45 (APC, BD, catalog 555485 or APC-Cy7, BD, catalog 557833); hCD3 (FITC, BD, catalog 555339); hCD4 (APC-H7, BD, catalog 560158); hCD8 (PerCP, BD, catalog 347314); hCD19 (PE-Cy7, BD, catalog 557835); hCD11b (PE, BD, catalog 555388 or APC, catalog 340937); hCD14 (FITC, BD, catalog 555397); hCD16 (PE-Cy7, BD, catalog 557744); and/or hCD33 (PE, BD, catalog 340679).

Techniques: Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: T cells establish and maintain CNS viral infection in HIV-infected humanized mice

doi: 10.1172/JCI98968

Figure Lengend Snippet: (A) The presence of human immune cells in the cerebrum (blue), brain stem (including midbrain, interbrain, and hindbrain, red), and cerebellum (green) of brains harvested from BLT mice was analyzed using IHC. A sagittal section of a BLT mouse brain was stained for hCD45. Scale bar: 1 mm. (B) Brain sections from BLT mice were stained with antibodies specific for human hematopoietic cells (hCD45), T cells (hCD3), and macrophages (hCD68). Scale bars: 50 μm. Original magnification, ×2 (insets). Positive cells are stained brown. CB, cerebellum; CC, cerebral cortex; CP, caudate putamen; HC, hippocampus; HT, hypothalamus; M, medulla; MB, midbrain, OB, olfactory bulb; P, pons; TH, thalamus; VS, ventral striatum.

Article Snippet: The antibody panel used to analyze cells isolated from humanized mice included antibodies directed against hCD45 (APC, BD, catalog 555485 or APC-Cy7, BD, catalog 557833); hCD3 (FITC, BD, catalog 555339); hCD4 (APC-H7, BD, catalog 560158); hCD8 (PerCP, BD, catalog 347314); hCD19 (PE-Cy7, BD, catalog 557835); hCD11b (PE, BD, catalog 555388 or APC, catalog 340937); hCD14 (FITC, BD, catalog 555397); hCD16 (PE-Cy7, BD, catalog 557744); and/or hCD33 (PE, BD, catalog 340679).

Techniques: Staining

Journal: The Journal of Clinical Investigation

Article Title: T cells establish and maintain CNS viral infection in HIV-infected humanized mice

doi: 10.1172/JCI98968

Figure Lengend Snippet: Total numbers of human (A) hematopoietic cells (hCD45+), (B) T cells, (C) CD4+ T cells, (D) CD8+ T cells, (E) the CD4+/CD8+ T cell ratio, and the numbers of (F) myeloid cells in the brains of HIV-infected (n = 132) or uninfected (n = 104, from Figure 3) BLT mice were determined by flow cytometry. Left panels indicate the absolute number of cells or CD4+/CD8+ T cell ratio in the brains of uninfected and HIV-infected BLT mice. Right panels indicate the absolute number of cells or CD4+/CD8+ T cell ratio in the brains of HIV-infected BLT mice and the time points after infection at which analysis was done. Mice infected with the same HIV strain are shown with the same color and symbol (CH040: green circles; JR-CSF: blue inverted triangles; RHPA: gray triangles; THRO: red diamonds; LAI: orange squares; ADA: black x). Horizontal lines indicate the mean ± SEM (A–F, left). *P < 0.05, **P < 0.01, and **** P < 0.0001, by Mann-Whitney U test for comparison of absolute cell numbers and CD4+/CD8+ T cell ratios in the brains of uninfected and HIV-infected BLT mice (A–F, left). The correlation over time for each parameter was assessed using a Spearman’s rank correlation test. P values are shown on each graph. The dashed gray lines on the plots on the right represent the mean values for the uninfected group.

Article Snippet: The antibody panel used to analyze cells isolated from humanized mice included antibodies directed against hCD45 (APC, BD, catalog 555485 or APC-Cy7, BD, catalog 557833); hCD3 (FITC, BD, catalog 555339); hCD4 (APC-H7, BD, catalog 560158); hCD8 (PerCP, BD, catalog 347314); hCD19 (PE-Cy7, BD, catalog 557835); hCD11b (PE, BD, catalog 555388 or APC, catalog 340937); hCD14 (FITC, BD, catalog 555397); hCD16 (PE-Cy7, BD, catalog 557744); and/or hCD33 (PE, BD, catalog 340679).

Techniques: Infection, Flow Cytometry, MANN-WHITNEY, Comparison

Journal: The Journal of Clinical Investigation

Article Title: T cells establish and maintain CNS viral infection in HIV-infected humanized mice

doi: 10.1172/JCI98968

Figure Lengend Snippet: (A) The numbers of human T cells in the brains of ToM were quantified using flow cytometry (n = 14), and the distribution of human cells was confirmed by staining brain sections with antibodies specific for hCD45, hCD3, hCD4, and hCD8 (positive cells are stained brown). (B) HIV RNA levels in the brains of ToM infected with T cell–tropic HIV-1 JR-CSF (n = 22). The dashed horizontal line represents the lower limit of detection for cell-associated RNA (~4 copies). The numbers of human (C) hematopoietic cells, (D) T cells, (E) CD4+ T cells, and (F) CD8+ T cells in the brains of uninfected (n = 14) and HIV-infected ToM (n = 26). (G) CD4+/CD8+ T cell ratio in the brains of uninfected (n = 14) and HIV-infected ToM (n = 26). A Mann-Whitney U test was used to analyze the data in C–G. *P < 0.05, **P < 0.01. Horizontal lines in A–G indicate the mean ± SEM. (H) Brain tissue sections from a systemically infected ToM were stained for HIV p24+ cells (brown). Scale bars: 50 μm. Original magnification, ×2 (insets).

Article Snippet: The antibody panel used to analyze cells isolated from humanized mice included antibodies directed against hCD45 (APC, BD, catalog 555485 or APC-Cy7, BD, catalog 557833); hCD3 (FITC, BD, catalog 555339); hCD4 (APC-H7, BD, catalog 560158); hCD8 (PerCP, BD, catalog 347314); hCD19 (PE-Cy7, BD, catalog 557835); hCD11b (PE, BD, catalog 555388 or APC, catalog 340937); hCD14 (FITC, BD, catalog 555397); hCD16 (PE-Cy7, BD, catalog 557744); and/or hCD33 (PE, BD, catalog 340679).

Techniques: Flow Cytometry, Staining, Infection, MANN-WHITNEY

Journal: Cellular and Molecular Immunology

Article Title: Human dNK cell function is differentially regulated by extrinsic cellular engagement and intrinsic activating receptors in first and second trimester pregnancy

doi: 10.1038/cmi.2015.66

Figure Lengend Snippet: Quantification of the dNK cell population throughout the first to second trimester of pregnancy. (a) Decidual leukocytes were isolated from the 6- to 20-week pregnancy period. The percentage of viable CD45+CD56+CD16− dNK cells at different gestational ages was illustrated in a scatterplot. Regression trend lines and R2 value are included. n = 65. (b) Histograms summarize the average proportion of first and second trimester dNK cells based on flow cytometric results. n = 34 (first trimester; 9 ± 1.9 week) or n = 31(second trimester; 16 ± 1.5 week). (c) Representative photographs of CD45 and CD56 immunohistochemical staining of serial sections of first and second trimester decidual tissues. High power images were inserted. Scale bar = 100 μm. (d) Summary data of the CD56+ dNK cell proportion amongst the CD45+ leukocytes from immunohistological analysis. n = 19 (first trimester) and 18 (second trimester).

Article Snippet: After non-specific blocking with serum-free protein block (Dako, Glostrup, Denmark), the cells were stained with following antibodies for 30 minutes at 4 °C to investigate their dNK phenotype: mouse anti-human CD45-APC/Cy7, CD56-PE/Cy7, CD3-Alexa Fluor 700 (BD Pharmingen, San Jose, CA, USA) and CD16-Krome Orangeplus combinations of either NKp30/CD337-PE, NKp46/CD335-PE/Cy5, and NKp44/CD336-Alexa647 or NKp80-PE, 2B4/CD244-PE/Cy5, and NKG2D/CD314-APC (Beckman Coulter, San Jose, CA, USA).

Techniques: Isolation, Immunohistochemical staining, Staining

Journal: Cellular and Molecular Immunology

Article Title: Human dNK cell function is differentially regulated by extrinsic cellular engagement and intrinsic activating receptors in first and second trimester pregnancy

doi: 10.1038/cmi.2015.66

Figure Lengend Snippet: NKp80 and NKG2D expression levels were upregulated on second trimester dNK cells. (a) Representative histograms of multiple dNK cell activation marker expression from the 8th (first trimester; solid line) and 14th (second trimester; dotted line) week deciduae. Viable CD45+CD56+CD16− dNK cells were gated. A fluorescence minus one (FMO) control is shown in gray. (b) NKp46, NKp44, NKp30, 2B4, NKG2D, and NKp80 expression on dNK cells from 6- to 20-week decidual samples (n = 65). (c) The average percentage of these activation markers between the first and second trimester dNK cells. n = 34 (first trimester; 9 ± 1.9 week) or n = 31 (second trimester; 16 ± 1.5 week). *p < 0.05.

Article Snippet: After non-specific blocking with serum-free protein block (Dako, Glostrup, Denmark), the cells were stained with following antibodies for 30 minutes at 4 °C to investigate their dNK phenotype: mouse anti-human CD45-APC/Cy7, CD56-PE/Cy7, CD3-Alexa Fluor 700 (BD Pharmingen, San Jose, CA, USA) and CD16-Krome Orangeplus combinations of either NKp30/CD337-PE, NKp46/CD335-PE/Cy5, and NKp44/CD336-Alexa647 or NKp80-PE, 2B4/CD244-PE/Cy5, and NKG2D/CD314-APC (Beckman Coulter, San Jose, CA, USA).

Techniques: Expressing, Activation Assay, Marker, Fluorescence

Journal: Cellular and Molecular Immunology

Article Title: Human dNK cell function is differentially regulated by extrinsic cellular engagement and intrinsic activating receptors in first and second trimester pregnancy

doi: 10.1038/cmi.2015.66

Figure Lengend Snippet: Functional changes of the dNK subsets at different gestation stage. (a) Representative dot plots of IFN-γ and CD107a expression by dNK cells from 10.5 and 15.4 weeks of gestation. Decidual leukocytes were cultured for 4 hours with a cell stimulation cocktail and CD107a antibody, then the cells were collected and processed with intracellular IFN-γ staining. Viable CD45+CD56+CD16− dNK cells were gated and studied. (b) Percentages of IFN-γ, CD107a positive dNK cells at different gestational stages of pregnancy. n = 18 (first trimester; 9 ± 1.6 week) or n = 12 (second trimester; 16 ± 1.3 week). The representative plots were from a 14th week decidua. (c) IFN-γ and CD107a expression of different dNK subsets based on their NKp80, NKG2D profile. *p < 0.05.

Article Snippet: After non-specific blocking with serum-free protein block (Dako, Glostrup, Denmark), the cells were stained with following antibodies for 30 minutes at 4 °C to investigate their dNK phenotype: mouse anti-human CD45-APC/Cy7, CD56-PE/Cy7, CD3-Alexa Fluor 700 (BD Pharmingen, San Jose, CA, USA) and CD16-Krome Orangeplus combinations of either NKp30/CD337-PE, NKp46/CD335-PE/Cy5, and NKp44/CD336-Alexa647 or NKp80-PE, 2B4/CD244-PE/Cy5, and NKG2D/CD314-APC (Beckman Coulter, San Jose, CA, USA).

Techniques: Functional Assay, Expressing, Cell Culture, Cell Stimulation, Staining

Journal: Cellular and Molecular Immunology

Article Title: Human dNK cell function is differentially regulated by extrinsic cellular engagement and intrinsic activating receptors in first and second trimester pregnancy

doi: 10.1038/cmi.2015.66

Figure Lengend Snippet: Immunological staining of dNK cells and trophoblast during the first and second pregnancy trimesters. (a) Representative immunohistochemistry of CD45, CD56, HLA-G, and CK7 to visualize the localization of CD45+ decidual leukocytes, CD56+ dNK and HLA-G+ EVT, CK7+ trophoblasts. (b) Co-staining images of dNK (CD56+; green) and trophoblast (CK7+; red) cells. Scale bar = 100 μm. n = 22 (first trimester; 9 ± 2.0 week) or n = 14 (second trimester; 15 ± 1.7 week).

Article Snippet: After non-specific blocking with serum-free protein block (Dako, Glostrup, Denmark), the cells were stained with following antibodies for 30 minutes at 4 °C to investigate their dNK phenotype: mouse anti-human CD45-APC/Cy7, CD56-PE/Cy7, CD3-Alexa Fluor 700 (BD Pharmingen, San Jose, CA, USA) and CD16-Krome Orangeplus combinations of either NKp30/CD337-PE, NKp46/CD335-PE/Cy5, and NKp44/CD336-Alexa647 or NKp80-PE, 2B4/CD244-PE/Cy5, and NKG2D/CD314-APC (Beckman Coulter, San Jose, CA, USA).

Techniques: Staining, Immunohistochemistry

Journal: Cellular and Molecular Immunology

Article Title: Human dNK cell function is differentially regulated by extrinsic cellular engagement and intrinsic activating receptors in first and second trimester pregnancy

doi: 10.1038/cmi.2015.66

Figure Lengend Snippet: Trophoblast engagement drives dNK survival and proliferation. Decidual leukocytes were stained with CFSE and cultured for 6 days in complete RPMI 1640 medium with 10% FBS in the presence of (i) trophoblastic HTR-8 cells; (ii) HTR-8 CM; (iii) cytokine mixture of rhIL-2 (2 ng mL−1) and rhIL-15 (10 ng mL−1). No additional stimulatory cytokines were added to the HTR-8 or CM groups. Viable CD45+CD56+ dNK cells were gated, and their proliferation was assessed. n = 10 with average an gestational age of 11 ± 2.7 week.

Article Snippet: After non-specific blocking with serum-free protein block (Dako, Glostrup, Denmark), the cells were stained with following antibodies for 30 minutes at 4 °C to investigate their dNK phenotype: mouse anti-human CD45-APC/Cy7, CD56-PE/Cy7, CD3-Alexa Fluor 700 (BD Pharmingen, San Jose, CA, USA) and CD16-Krome Orangeplus combinations of either NKp30/CD337-PE, NKp46/CD335-PE/Cy5, and NKp44/CD336-Alexa647 or NKp80-PE, 2B4/CD244-PE/Cy5, and NKG2D/CD314-APC (Beckman Coulter, San Jose, CA, USA).

Techniques: Staining, Cell Culture

Journal: PLOS ONE

Article Title: Imiquimod induces skin inflammation in humanized BRGSF mice with limited human immune cell activity

doi: 10.1371/journal.pone.0281005

Figure Lengend Snippet: (a) Percent and (b) number of human γδTCR + of human CD45 + cells and (c) percent and (d) number of human pDCs of human CD45 + cells were analyzed in spleen cell suspensions by flow cytometry at terminations. Percent and (e) number (f) of human monocytes of human CD45 + cells were analyzed by flow cytometry on blood at terminations.

Article Snippet: Subsequently, blood samples were stained with live/dead stain (APC-R700, cat. 564997, BD Biosciences), mouse CD45 (BV786, clone 30-F11, cat. 564225, BD Biosciences) and human CD45 (APC-Cy7, clone 2DL, cat. 557833, BD Biosciences), CD14 (APC, clone M5E2, cat. 561708, BD Biosciences), CD15 (BV711, clone W6D3, cat. 563142, BD Biosciences) and CD16 (BV605, clone 3G8, cat. 563173, BD Biosciences) diluted in a mixture of FACS buffer and Brilliant Stain Buffer Plus (cat. 566385, BD Biosciences).

Techniques: Flow Cytometry