Journal: Journal of Neuroscience

Article Title: Vascular Remodeling versus Amyloid -Induced Oxidative Stress in the Cerebrovascular Dysfunctions Associated with Alzheimer's Disease

doi: 10.1523/jneurosci.4031-05.2005

Figure Lengend Snippet: Figure6. NitrosativestressinthecerebralcortexofAPP andTGF mice.Nitrotyrosineimmunoreactivityisupregulatedin the vessel walls of young APP mice, whereas it is distributed in the neuropil and around intraparenchymal blood vessels in 12-month-old APP mice. As shown in the adjacent panels, nitrotyrosine immunoreactivity (red) is mainly located in CD11b- immunopositive microglial processes (green; the merge with nitrotyrosine is yellow) and cells (open arrows), but some neurons are also nitrotyrosine immunopositive (small arrows). In TGF mice, nitrotyrosine immunoreactivity is primarily distributed to glial elements in both wild-type (WT) and transgenic mice. Scale bar, 75 m. mo, Month.

Article Snippet: Changes in pial and/or intracortical microvessels in transgenic mice (perfusion-fixed brains) and AD patients (50- m-thick sections) were assessed by immunostaining for the following markers: SOD2 (rabbit anti-MnSOD; 1:400; Stressgen), nitrosative stress (mouse anti-nitrotyrosine; 1:1000; Upstate Biotechnology, Lake Placid, NY), basement membrane proteins collagen-I (goat anti- collagen type I; 1:400; Southern Biotechnology, Birmingham, AL) and collagen-IV (goat anti-collagen type IV; 1: 400; Chemicon), and astroglial (GFAP, rabbit anti-GFAP; 1:300; Dako, Glostrup, Denmark) or microglial (rat anti-CD11b; 1:100; Serotec, Raleigh, NC) activation.

Techniques: Transgenic Assay

Journal: Journal of the American Heart Association

Article Title: Transplantation of Allogeneic Pericytes Improves Myocardial Vascularization and Reduces Interstitial Fibrosis in a Swine Model of Reperfused Acute Myocardial Infarction

doi: 10.1161/jaha.117.006727

Figure Lengend Snippet: Figure 6. Flow cytometry analyses of APCs isolated from human and swine saphenous veins. A and B, Representative flow cytometry gating procedure of hAPC line #1 and sAPC line #1 at P5. Total cell populations and the single cells (singlets) were gated according to FSC-A vs SSC-A and FSC-A vs FSC-H parameters (i and ii). Viable cells were distinguished from dead cells using Fixable Viability Dye eFluor780 (iii) and further gated for selected antigens (iv through xi and iv through xiii). Pericyte, mesenchymal, endothelial, and hematopoietic markers were studied. The FMO control was used in the assessment and gating of CD146+ and PDGFRb+ cells, because of the use of multiple fluorochromes (vii through ix and viii through x). The same approach was used when studying the expression of CD45 and CD11b on sAPCs to exclude hematopoietic cell contamination in the cell culture system (xi through xiii). Data were acquired using FACSCantoII (BD Biosciences) or Novocyte 3000 flow cytometer (ACEA Biosciences, San Diego, CA, USA) and analyzed using the FlowJo v10.3 software. C and D, Flow cytometry histograms for each surface marker in representative hAPC (C) and sAPC lines (D). Negative control staining profiles are shown by the red histograms, whereas specific antibody staining profiles are shown by light blue histograms. Bar graphs show the meanSEM values of 3 hAPC and sAPC lines. E and F, Gating and histograms of fresh isolated swine PB-MNCs and swine PAEC line #1 at P5 used as positive control for the staining of hematopoietic and endothelial markers, respectively. In both cell lines, the negative control staining profile is shown by full red histogram, while the positive staining profile is shown by full light-blue histogram. FSC-A indicates Forward Scatter Area; FSC-H, Forward Scatter Height; FMO, fluorescence minus 1; hAPCs, human adventitial pericytes; PB-MNCs, Peripheral blood mononuclear cells; PDGFRb, platelet-derived growth factor receptor-b; sAPCs, swine adventitial pericytes.

Article Snippet: Antibodies used in flow cytometry studies on sAPCs Marker Permeabilization Reactivity Primary antibody source dilution Fluorophores CD90 No Swine eBioscience, 1:20 Pe-Cy7 CD44 No Swine eBioscience, 1:20 APC CD105 No Swine LifeSpan, 1:5 PE CD45 No Swine Serotec, 1:25 FITC CD11b No Human/Swine eBioscience, 1:20 Pe-Cy7 CD31 No Swine Serotec, 1:80 PE CD146 No Swine Serotec, 1:60 FITC PDGFR No Human Biolegend, 1:25 PE Dye eFluor 780 No eBioscience, 1:1000 APC-Cy7 Cells (1x106, N=3 biological replicates) were washed in 1X DPBS (Life Technologies) and treated with 10X trypsin/EDTA (Life Technologies).

Techniques: Flow Cytometry, Isolation, Cytometry, Control, Expressing, Cell Culture, Software, Marker, Negative Control, Staining, Positive Control, Derivative Assay

Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: Myeloid cells dominate the acute response to ICH and contribute to early damage. (A) Single cells were isolated from the right hemisphere of naïve, sham-operated and collagenase-induced ICH mice 24 h post-surgery and immunophenotyped using flow cytometry. (B) Left: gating strategy used during flow cytometry analysis. Right: cell counts are shown from the following populations: CD45 int CD11b + microglia, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ), CD45 hi CD11b − CD45R + B cells, and CD45 hi CD11b − CD3 + T cells. Data presented as mean+s.e.m., n =5, two independent experiments. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001, **** P <0.0001, determined by one-way ANOVA with Tukey's post-hoc test. (C) Formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (left) and ICH (right) cases were immunostained for the myeloid cell marker CD11b (green) and DAPI (blue), with red blood cell (RBC) autofluorescence shown in red. One representative image from three cases per group shown. Scale bars: 100 µm (50 μm in insets). (D) White blood cells were isolated from whole blood of wild-type (WT) ( n =7) and chemokine receptor 2 ( Ccr2 ) −/− ( n =9) male littermate mice. Top left: CD115 + monocytes and Ly6G + neutrophils as a percentage of all CD45 + immune cells. Bottom left: representative flow plots of the circulating monocyte compartment of each genotype. Right: the amount of Ly6C hi monocytes as a percentage of total circulating monocytes from each genotype. ns, not significant; **** P <0.0001, determined by unpaired two-tailed t -test. (E) Following a 3 day training period on the rotarod assay, WT and Ccr2 −/− littermates were subjected to collagenase-induced ICH and tested on the rotarod again 24 h later. Data presented as net difference in latency to fall from baseline performance, n =7-9, four independent experiments. * P <0.05, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Isolation, Flow Cytometry, Formalin-fixed Paraffin-Embedded, Control, Marker, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: IL-1 produced by mononuclear phagocytes controls acute recruitment of myeloid cells during ICH. (A) Mice were subjected to collagenase-induced ICH and culled at 4 h ( n =5) and 24 h ( n =4) post-ICH. 20 µm coronal brain sections were immunostained for either IL-1α (green, top row) or IL-1β (green, bottom row) together with Iba1 (red) and DAPI (blue). Dashed lines represent haematoma border, arrows define haematomal IL-1β+ cells, and insets are higher-magnification images of respective white boxes. Scale bars: 50 µm. (B) Quantification of IL-1 + cells. (C) 8 µm sections of formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (top) and acute ICH (bottom) cases were immunostained for Iba1 (red), IL-1β (green) and DAPI (blue). Red blood cell (RBC) autofluorescence can be seen in white. One representative image from three patients per group is shown. Scale bars: 100 µm. Insets are higher-magnification images of the areas within the white boxes. (D) Representative flow plot of cells isolated from brains of mice injected with central (10 µg intrastriatal) and peripheral (100 mg kg −1 subcutaneous) IL-1 receptor antagonist (IL-1Ra) ( n =4) or placebo ( n =5) and subjected to ICH, two independent experiments. (E) Cell counts are shown from the following populations: CD45 hi CD11b − lymphocytes, CD45 int CD11b + microglia, CD45 hi CD11b + myeloid cells, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ). Data presented as mean+s.e.m. ns, not significant; * P <0.05, ** P <0.01, *** P <0.001, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Produced, Formalin-fixed Paraffin-Embedded, Control, Isolation, Injection, Two Tailed Test