Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet: Endothelial, fibroblast and cardiomyocyte cell purification from mouse hearts (A) Scheme on the experimental procedures to isolate the indicated cell types from mouse hearts. (B) Heatmap of cell marker gene expression in RNA from isolated cardiomyocytes (CM), endothelial cells (EC) or fibroblasts (FB) after sham or the indicated time point after TAC surgery. (C) Cardiac endothelial cells and fibroblasts were isolated from mouse hearts and stained for the endothelial markers CD31 and CD102, for the leukocyte marker CD45, and the fibroblast marker Mefsk4. Subsequently, flow cytometric analyses were performed and representative results are shown here. The numbers indicated in each quadrant indicates the percentage of cells localized in that particular quadrant. (D) RNA from the different cell types after sham or 1 and 8 weeks after TAC was subjected to RNA sequencing. The differences in overall gene expression patterns were visualized by a principal component analysis.

Article Snippet: CD45 Antibody, anti-mouse , Miltenyi Biotec , Clone 30F11.

Techniques: Purification, Marker, Gene Expression, Isolation, Staining, RNA Sequencing

Journal: iScience

Article Title: Analysis of myocardial cellular gene expression during pressure overload reveals matrix based functional intercellular communication

doi: 10.1016/j.isci.2022.103965

Figure Lengend Snippet:

Article Snippet: CD45 Antibody, anti-mouse , Miltenyi Biotec , Clone 30F11.

Techniques: Purification, Plasmid Preparation, Blocking Assay, Magnetic Beads, Recombinant, Clinical Proteomics, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: eLife

Article Title: The transcriptomic and epigenetic map of vascular quiescence in the continuous lung endothelium

doi: 10.7554/eLife.34423

Figure Lengend Snippet: ( A ) Sorting strategy for lung EC isolation. PTPRC ‐ LYVE1 ‐ LY76 ‐ PDPN ‐ PI ‐ CD31 + CD34 + cells were used for transcriptome and methylome analysis. Propidium iodide (PI) was used to discriminate between live (PI ‐ ) and dead (PI + ) cells. ( B ) Validation of the sorted EC populations by marker gene expression analysis using qPCR. n = 3; mean ± SD. ( C ) Size analysis of EC populations (CD31 + CD34 + ) isolated from infant and young adult mice. ( D ) Analysis of EC proliferation from brain, kidney, liver and heart by Ki67 and Cd31 co-staining. n ≥ 5; mean ± SD. ( E ) Validation of the differential expression of cell cycle genes in infant and young adult brain and heart EC using qPCR. n ≥ 3; mean ± SD; *p<0.05; Mann-Whitney Test.

Article Snippet: PTPRC ‐ LYVE1 ‐ LY76 ‐ PDPN ‐ PI ‐ CD31 + CD34 + cells were sorted with a BD FACSAria ll (BD Biosciences, Heidelberg, Germany) The following primary antibodies were used: rat anti-CD31 (551262, BD Biosciences, diluted 1:100), rat anti-CD34 (48–0341, eBioscience, diluted 1:50), rat anti-LY76 (561032, BD Biosciences, diluted 1:200), rat anti-LYVE1 (53–0443, eBioscience, diluted 1:250), hamster anti-PDPN (53–5381, eBioscience, 1:100), rat anti-PTPRC (553080, BD Biosciences, 1:400).

Techniques: Isolation, Marker, Expressing, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Targeting extracellular vesicle delivery to the lungs by microgel encapsulation

doi: 10.1101/2022.09.09.507125

Figure Lengend Snippet: HUVEC and macrophages were exposed to DiR labelled MSC-EVs in vitro . EV uptake by HUVEC was characterized after 24h by (A) flow cytometry and (B) AMNIS analysis revealing the internalization of EVs by a limited population of HUVEC, as quantified (C). Unstimulated macrophages (MØ), IL4 stimulated and LPS/IFNg stimulated macrophages readily took up macrophages as shown in representative flow plots (D) and quantified (E). Data represents mean ± SEM.

Article Snippet: Macrophages were characterized for presence and absence of canonical markers using flow cytometry PE-Cy5 mouse anti-rat CD45 (BD Biosciences, Mississauga, ON, Canada), BV786 mouse anti-rat CD11b/c (BD Biosciences, Mississauga, ON, Canada), and PE mouse anti-rat CD34 (Novus Biologicals, Littleton, CO, USA). ( Supplemental Figure 2 ).

Techniques: In Vitro, Flow Cytometry

Journal: bioRxiv

Article Title: Targeting extracellular vesicle delivery to the lungs by microgel encapsulation

doi: 10.1101/2022.09.09.507125

Figure Lengend Snippet: Lungs were isolated, digested, and prepared for flow cytometry analysis 24h after DiR labelled EV administration. (A) Representative flow plots demonstrate consistent proportions of lung endothelial (CD45-CD31+), immune (CD45+CD31- or CD31lo), and other (CD45-CD31-) populations, which was quantified as cell counts, and proportions (B) demonstrating increased numbers of immune cells with the presence of microgels. EV uptake was assessed by evaluation of DiR labelling, representative flow plots are shown (C) and quantification (D) demonstrates significant EV uptake by CD45+CD31- and CD45+CD31lo cells when exposed to encapsulated EVs. Data represented as mean ± SEM, * p<0.05 PBS, Free EVs, and Empty Capsules compared to Encap. EVs, *** p<0.0005 all Tx compared to Encap EVs, # p = 0.07 healthy compared to Encap EVs.

Article Snippet: Macrophages were characterized for presence and absence of canonical markers using flow cytometry PE-Cy5 mouse anti-rat CD45 (BD Biosciences, Mississauga, ON, Canada), BV786 mouse anti-rat CD11b/c (BD Biosciences, Mississauga, ON, Canada), and PE mouse anti-rat CD34 (Novus Biologicals, Littleton, CO, USA). ( Supplemental Figure 2 ).

Techniques: Isolation, Flow Cytometry

Journal: European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery

Article Title: Decellularized mitral valve in a long-term sheep model.

doi: 10.1093/ejcts/ezx485

Figure Lengend Snippet: Figure 7: Immunostaining of the decellularized mitral valve (DMV) and the biological mitral valve (BMV). (A and B) DMV and (C and D) BMV. (A, C) CD45 and DNA; (B, D) procollagen I and DNA. DMV shows collagen-producing cells within the leaflets. BMV has almost no cells, positive for procollagen I. The CD45-positive cells in the BMV tend to form nodules (picture of the region, close to the annulus). BMV has only several CD45-positive cells on the surface. Ao: Aorta ascendens.

Article Snippet: D ow nloaded from https://academ ic.oup.com /ejcts/article/53/6/1165/4828177 by guest on 22 M ay 2024 • Collagen IV (clone CI22, DAKO, Hamburg, Germany) • Collagen I C2345 clone COL-I (Sigma-Aldrich Chemie GmbH, Munich, Germany) • CD31 [monoclonal mouse immunoglobulin G (IgG)2a, Serotec, Raleigh, NC, USA] • von Willebrand factor (polyclonal rabbit IgG, DAKO) • Endothelial nitric oxide synthase (monoclonal mouse IgG1, eNOS/NOS Type III, BD Transduction Laboratories, San Jose, CA, USA) • CD45 [mouse monoclonal (clone OX-1) IgG, BioRad, Oxford, UK] • Procollagen (monoclonal mouse IgG1, M-38-c, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) The images were obtained using the microscope AxioObserver A1, with the camera AxioCamMRm (Carl Zeiss, Jena, Germany).

Techniques: Immunostaining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Phenotypic miRNA Screen Identifies miR-26b to Promote the Growth and Survival of Endothelial Cells

doi: 10.1016/j.omtn.2018.08.006

Figure Lengend Snippet: Time Course of Endogenous miR-26b Expression in the Adductor Muscle following Ischemia Injury (A and B) Expression of miR-26b in (A) endothelial cell (CD31 pos /CD45 neg ) fraction and (B) muscle fraction isolated from the adductor muscles, which were collected at 3, 7, and 14 days after ischemia induction. miR-26b levels were normalized against snRU6 control. Error bars are mean ± SEM; **p < 0.01 and ***p < 0.001 compared to controls (n = 6 replicates, unpaired t test).

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody rat anti-mouse CD45 (Pharmagen; 1:100) overnight at 4°C.

Techniques: Expressing, Isolation, Muscles, Control

Journal: Molecular Therapy. Nucleic Acids

Article Title: Phenotypic miRNA Screen Identifies miR-26b to Promote the Growth and Survival of Endothelial Cells

doi: 10.1016/j.omtn.2018.08.006

Figure Lengend Snippet: miR-26b Mimic Preserves the Native Microvasculature Network Morphology following Ischemia Injury Representative confocal microscopy images of thick longitudinal sections for the ischemic adductor muscles injected with control mimic (left panels) or miR-26b mimic (right panels) at day 3 after ischemia. Upper panel: effect of exogenous miR-26b mimic injection on the survival of native muscle fibers and microvasculature network is shown. Asterisks denote the necrotic (dead) muscle fibers. Dotted line delimits the border of necrotic area. Myocytes (phalloidin, green), microvessels (isolectinB4, red), and nuclei (DAPI, blue) are shown. Scale bar, 340 μm. Middle panel: effect of exogenous miR-26b mimic injection on the native microvasculature network and the infiltration of leukocytes in the ischemic muscles is shown. Leukocytes (CD45, green) and microvessels (isolectinB4, red) are shown. Scale bar, 340 μm. Lower panel: inset of a higher magnification shows the microvascular network architecture in the necrotic area of ischemic adductor muscle. Arrowheads point to the preserved native microvasculature network in the necrotic areas. Leukocytes (CD45, green), microvessels (isolectinB4, red), and nuclei (DAPI, blue) are shown. Scale bar, 80 μm.

Article Snippet: Briefly, following incubation with blocking buffer, the samples were incubated with primary antibody rat anti-mouse CD45 (Pharmagen; 1:100) overnight at 4°C.

Techniques: Confocal Microscopy, Muscles, Injection, Control