Journal: Science Advances

Article Title: Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration

doi: 10.1126/sciadv.add1101

Figure Lengend Snippet: ( A ) Representative dot plots showing CD45 int CD11b + microglia with AF low (AF lo ) and AF high (AF hi ) properties in young and old mice, respectively. ( B ) Microglia counts in young and old brain hemispheres, including for each AF subset, are shown. ( C ) The level of AF in each bulk microglial population and subset is quantified using the mean fluorescence intensity (MFI) of the empty fluorescein isothiocyanate (FITC) channel. Representative histograms are shown next to the relative MFI quantification of ( D ) lipid content, ( E ) lipid peroxidation levels, and ( F ) iron accumulation in microglia as demonstrated using fluorogenic dyes. Phagocytic activity was measured by ( G ) intracellular detection of the neuronal marker MAP2, ( H ) fluorescence of neuronal SLICK-YFP (single-neuron labeling with inducible Cre-mediated knockout–yellow fluorescent protein) reporter mice, and ( I ) intracellular detection of the myelin marker, myelin CNPase. ( J and K ) Oxidative stress was measured by intracellular protein expression of NOX2 and production of ROS using dihydrorhodamine (DHR) 123. N = 5 per group. Fluorescence minus one (FMO) controls are shown in gray, while microglial populations are color-coded according to bar graph. A vertical fiducial line is included for reference. AF, autofluorescence; Max, maximum; Ox, oxidation; Red, reduction; SSC, side scatter; ns, not significant. Nonparametric data were analyzed using Mann-Whitney test (* P < 0.05 and ** P < 0.01).

Article Snippet: Surface antibody cocktails were prepared with CD45/PerCP-Cy5.5 (1:100; BioLegend) and CD11b/APC-Fire 750 (1:100; BioLegend) including viability dye Zombie Aqua.

Techniques: Fluorescence, Activity Assay, Marker, Labeling, Knock-Out, Expressing, MANN-WHITNEY

Journal: Science Advances

Article Title: Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration

doi: 10.1126/sciadv.add1101

Figure Lengend Snippet: ( A ) Representative dot plots show the immune profile in the brains of young and middle-aged Apoe , h APOE3 , and h APOE4 , quantified on the right. Representative histograms depict the relative level of cell granularity ( B ), autofluorescence ( C ), lipid accumulation ( D ), CD68 ( E ), and Lamp1 ( F ) protein expression and intracellular cytokine production of TNF ( G ) in CD45 int CD11b + microglia across ages and genotypes. ( H ) Ex vivo neuronal engulfment assay shows a significant increase in the percent of microglia that phagocytized live SLICK-YFP neurons, quantified on the right. Validation of internalized myelinated cortical neurons was performed using intracellular detection of anti-YFP ( I ) and anti-myelin CNPase ( J ) in phagocytic (SLICK-YFP + ) and nonphagocytic (SLICK-YFP − ) microglial populations within the same brain. N = 6 to 7 per group. MA, middle-aged; Y, young; ns, not significant.. Data were analyzed using one-way analysis of variance (ANOVA) with Bonferroni post hoc correction for multiple comparisons (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: Surface antibody cocktails were prepared with CD45/PerCP-Cy5.5 (1:100; BioLegend) and CD11b/APC-Fire 750 (1:100; BioLegend) including viability dye Zombie Aqua.

Techniques: Expressing, Ex Vivo

Journal: Science Advances

Article Title: Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration

doi: 10.1126/sciadv.add1101

Figure Lengend Snippet: ( A ) Timeline of experimental design immediately before sham and TBI surgery. ( B ) Representative dot plots of immune populations in the ipsilateral brain hemisphere at 3 days after TBI. Quantification of CD45 int CD11b + microglia and CD45 hi CD11b + -infiltrating myeloid cell counts per hemisphere are shown for aged, surgery, and treatment groups. ( C ) A representative histogram of DHR123 + microglia is shown next to the relative MFI quantification of ROS production. In the associated histogram, young groups are shown with no outline, old groups are shown with bold outlines, and treatment groups are color-coded according to bar graph and figure legend (Veh in red and PLX5622 in blue). A vertical fiducial line is included for reference. Representative dot plots depicting ( D ) IL-1β production and ( E ) p16 expression in microglia are shown next to quantification of cell frequencies. ( F ) Cytokine protein concentrations in the peri-lesional cortex as measured by ELISA. No differences were seen between sham control groups after treatment, and so data for both sham groups were combined. For all cytokines (M-CSF, G-CSF, eotaxin, IL-1a, MIG, and MIP-2), TBI acutely increased concentrations in Veh but not PLX5622-treated groups. N = 3 to 4 per sham and 6 to 7 per TBI group. ns, not significant. Data were analyzed using two-way ANOVA with Tukey post hoc correction (B to E) and one-way ANOVA with Bonferroni post hoc correction (F) for multiple comparisons (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: Surface antibody cocktails were prepared with CD45/PerCP-Cy5.5 (1:100; BioLegend) and CD11b/APC-Fire 750 (1:100; BioLegend) including viability dye Zombie Aqua.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Science Advances

Article Title: Brain injury accelerates the onset of a reversible age-related microglial phenotype associated with inflammatory neurodegeneration

doi: 10.1126/sciadv.add1101

Figure Lengend Snippet: ( A ) Timeline of experimental design immediately before sham and TBI surgery. ( B ) Representative dot plots of immune populations in the ipsilateral and contralateral (internal control) brain hemispheres at 2 weeks after TBI. Quantification of CD45 int CD11b + microglia (left), infiltrating CD45 hi CD11b + myeloid (center), and CD45 hi CD11b − lymphocyte (right) counts per hemisphere is shown for aged, injury, and treatment groups. ( C ) A representative histogram of CD68 + microglia is shown next to the MFI quantification of this phagocytosis marker. ( D ) A representative histogram shows the relative level of neutral lipids in microglia from each hemisphere after TBI. ( E ) Representative dot plots depict the percentage of Lipi-Blue+ microglia after TBI. The frequency of lipid droplet–containing microglia is quantified. ( F ) A representative histogram shows the relative level of ROS production in microglia as measured by DCF probe. ( G ) Cytokine protein concentrations in the peri-lesional cortex as measured by ELISA. No differences were seen between sham control groups after treatment, and so data for both sham groups were combined. N = 3 to 4 per contralateral and 6 to 7 per ipsilateral (i.e., TBI) group. FMO controls are shown in gray, contralateral groups are shown with no outline, ipsilateral groups are shown with bold outlines, and treatment groups are color-coded according to bar graph and figure legend (TBI + Veh in red and TBI + PLX5622 in blue). Contra, contralateral; Ipsi, ipsilateral; ns, not significant.. Data were analyzed using two-way ANOVA with Tukey post hoc correction (B to F) and one-way ANOVA with Bonferroni post hoc correction (G) for multiple comparisons (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: Surface antibody cocktails were prepared with CD45/PerCP-Cy5.5 (1:100; BioLegend) and CD11b/APC-Fire 750 (1:100; BioLegend) including viability dye Zombie Aqua.

Techniques: Marker, Enzyme-linked Immunosorbent Assay

Journal: Breast Cancer Research : BCR

Article Title: Bone marrow-derived, alternatively activated macrophages enhance solid tumor growth and lung metastasis of mammary carcinoma cells in a Balb/C mouse orthotopic model

doi: 10.1186/bcr3195

Figure Lengend Snippet: M2-polarized macrophages enhance leukocyte infiltration into tumor tissues and monocyte migration in vitro . (A) 4T1 cells and/or M2-polarized macrophages (M2-Mϕs) were injected into the mammary fat pad of female Balb/C mice. M2-Mϕs were labeled with 3,3'-dioctadecyloxacarbocyanine perchlorate/DiOC 18 (DiO) (green) prior to the injection. Cryostat sections of tumor were stained with anti-CD45 antibody (red), and nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue). (B) 4T1 cells and/or M2-Mϕs were plated in 12-well plates, and 24-hour-conditioned media (CM) were collected for trans-well migration assays. The migration of bone marrow-derived monocytes was estimated using the CM as a chemoattractant. The migrated monocytes were quantified by counting the DAPI-stained cells. Each bar represents the mean ± standard error of the mean of three independent experiments performed in duplicate. Means without the same lowercase letter differ, P < 0.05.

Article Snippet: The following antibodies were purchased from the suppliers as indicated: antibodies against Ki-67, hypoxia-inducible factor-1 alpha (HIF-1α), and lymphatic vessel endothelial hyaluronan receptor-1 from Abcam (Cambridge, UK); antibodies against cyclin-dependent kinase (CDK)2, CDK4, vascular endothelial cell growth factor (VEGF)-A, VEGF-C, and CD31 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-CD45 antibody from R&D Systems (Minneapolis, MN, USA); antibodies against rabbit-IgG-Alexa488, rabbit-IgG-Alexa597, and goat-IgG-Alexa488 from Invitrogen (Carlsbad, CA, USA); and anti-goat-IgG-Cy3 antibody from Rockland (Gibertsville, PA, USA).

Techniques: Migration, In Vitro, Injection, Labeling, Staining, Derivative Assay

Journal: Cancer Discovery

Article Title: Aged and BRCA -Mutated Stromal Cells Drive Epithelial Cell Transformation

doi: 10.1158/2159-8290.CD-24-0805

Figure Lengend Snippet: hrMSCs are enriched in the stroma underlying and adjacent to fallopian tube STIC lesions. A, Representative H&E-stained tissue sections of normal fallopian tubes and fallopian tubes harboring STIC lesions. Histology is paired with multispectral IF images of each respective group shown. Black and white images denote cells with the phenotypes of interest. Red x’s indicate hrMSCs (WT1 + /CD73 + /CD90 + /CD105 + /CD45 − ), whereas yellow x’s indicate nMSCs (WT1−/CD73 + /CD90 + /CD105 + /CD45 − ). B, Schematic showing the criteria for the Vectra spatial quantification. C, hrMSC abundance normalized to the area of ROI. D, hrMSC to nMSC ratios. E, hrMSC abundance (normalized to the area of ROI) in the expanded normal FT patient cohorts consisting of WT ( n = 10 young, n = 9 aged), BRCA1 mutant ( n = 9), or BRCA2 mutant ( n = 6) fallopian tubes lacking STIC/HGSOC. F, Ratio of hrMSC/nMSC in normal FTs. P values were determined by ordinary one-way ANOVA with Tukey’s multiple comparisons analysis. Data points for C – F are reflective of individual ROIs. Statistics were determined on field ROIs for C and D . Statistics were determined with patient averages for E and F . G, Heatmap with unsupervised clustering of STIC stroma vs. normal stroma. H, Enrichment score of DEGs between STIC stroma vs. normal stroma (left: all, right: top 30 genes) applied to stromal location [from normal to STIC distal (dist) to STIC adjacent that contains both STIC contiguous and adjacent regions (STIC adj) to directly underlying STIC to invasive stroma]. I, Volcano plot of significantly differentially expressed genes in STIC stroma vs. normal stroma. J, Enrichment score of WT1 targets in stroma applied to stromal locations as in I . K, Correlation of stromal WT1 enrichment score with epithelial WT1 enrichment score.

Article Snippet: Anti-human antibodies used included: WT1 (Santa Cruz Biotechnology, Cat# sc-393498, RRID:AB_2905496), CD73 (Abcam, Cat# ab133582, RRID:AB_3674653), CD105 (Abcam, Cat# ab114052, RRID:AB_10900113), CD45 (Atlas Antibodies, Cat# AMAb90519, RRID:AB_2665572), CD90 (Novus, Cat# NBP1-43379G, RRID:AB_3209185), and panCK (Santa Cruz Biotechnology, Cat# sc-81714, RRID:AB_2191222).

Techniques: Staining, Mutagenesis

Journal: Scientific Reports

Article Title: c-Jun N-terminal kinase 1 defective CD4+CD25+FoxP3+ cells prolong islet allograft survival in diabetic mice

doi: 10.1038/s41598-018-21477-9

Figure Lengend Snippet: Lack of JNK1 expression enhances survival of Tregs in recipient mice. Pancreatic islets and Tregs from WT or JNK1 −/− mice (CD45.2) were transferred to diabetic mice (CD45.1) as in Fig. . Three, thirty and one hundred twenty days after transplantation, the percentages of donor-derived (CD45.2) cells in recipient mouse liver were determined by flow cytometry. (a) Schematic representation of the experiment. ( b ) Absolute number of cells. ( c ) A representative flow cytometry figure. Data presented are representative of five independent experiments. Five mice per group were used. Mean values, p values and SEs are shown.

Article Snippet: For insulin staining in the transplanted liver, monoclonal mouse primary anti-insulin (Abcam) and CD45.2 monoclonal mouse FITC-tagged anti-CD45.2 (Rockland Immunochemicals, Inc.) antibodies were used.

Techniques: Expressing, Transplantation Assay, Derivative Assay, Flow Cytometry